Genes encoding novel proteins with pesticidal activity against coleopterans

ABSTRACT

The invention provides nucleic acids, and variants and fragments thereof, obtained from strains of  Bacillus thuringiensis  encoding δ-endotoxins having pesticidal activity against pests of the order Coleoptera. The invention further provides mutagenized nucleic acids that have been modified to encode endotoxins having improved pesticidal activity and/or altered pest specificity. Particular embodiments of the invention provide isolated nucleic acids encoding pesticidal proteins, pesticidal compositions, expression cassettes, and transformed microorganisms and plants comprising a nucleic acid of the invention. These compositions find use in methods for controlling pests, especially plant pests.

CROSS-REFERENCE PARAGRAPH

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/242,838, filed Oct. 24, 2000, the teachings of whichare herein incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to naturally occurring andrecombinant nucleic acids obtained from Bacillus thuringiensis Cry8-likegenes that encode δ-endotoxins characterized by pesticidal activityagainst pests of the order Coleopteran. Compositions and methods of theinvention utilize the disclosed nucleic acids, and their encodedpesticidal polypetides, to control plant pests.

BACKGROUND OF THE INVENTION

[0003] Insect pests are a major factor in the loss of the world'sagricultural crops. Insect pest-related crop loss from corn rootwormalone has reached one billion dollars a year. For example, corn rootwormfeeding can be economically devastating to agricultural producers. Thewestern corn rootworm is a major insect pest of corn or maize in manyregions of the world. While not as important a pest as the western cornrootworm, the southern corn rootworm may occasionally cause significanteconomic damage to corn. Damage from western and southern corn rootwormsmay result in increased lodging, reduced drought tolerance andultimately, crop yield reductions.

[0004] Traditionally, the primary methods for impacting corn rootwormpopulations are crop rotation and the application of broad-spectrumchemical insecticides. Unfortunately, some species of pests havedeveloped resistance to the chemical insecticides. Furthermore,consumers and government regulators alike are becoming increasinglyconcerned with the environmental hazards associated with the productionand use of synthetic chemical pesticides. Because of such concerns,regulators have banned or limited the use of some of the more hazardouspesticides. Thus, there is substantial interest in developingalternative pesticides.

[0005] Biological control of insect pests of agricultural significanceusing a microbial agent, such as fungi, bacteria, or another species ofinsect affords an environmentally friendly and commercially attractivealternative. Generally speaking, the use of biopesticides presents alower risk of pollution and environmental hazards, and they providegreater target specificity than is characteristic of traditionalbroad-spectrum chemical insecticides. In addition, biopesticides oftencost less to produce and thus improve economic yield for a wide varietyof crops.

[0006] Certain species of microorganims of the genus Bacillus are knownto possess pesticidal activity against a broad range of insect pestsincluding Lepidoptera, Diptera, Coleoptera, Hemiptera, and others.Bacillus thuringiensis and Bacillus papilliae are among the mostsuccessful biocontrol agents discovered to date. Insect pathogenicityhas also been attributed to strains of: B. larvae, B. lentimorbus, B.papilliae, B. sphaericus, B. thuringiensis (Harwook, ed., ((1989)Bacillus Plenum Press), 306) and B. cereus (WO 96/10083). Pesticidalactivity appears to be concentrated in parasporal crystalline proteininclusions, and several genes encoding these pesticidal proteins havebeen isolated and characterized (see, for example U.S. Pat. No.5,366,892).

[0007] Microbial insecticides, particularly those obtained from Bacillusstrains, have played an important role in agriculture as alternatives tochemical pest control. Recently, agricultural scientists have developedcrop plants with enhanced insect resistance by genetically engineeringcrop plants to produce pesticidal proteins from Bacillus. For example,corn and cotton plants genetically engineered to produce pesticidalproteins isolated from strains of B. thuringiensis, known asδ-endotoxins or Cry toxins, are now widely used in American agricultureand have provided the farmer with an environmentally friendlyalternative to traditional insect-control methods. In addition, potatoesgenetically engineered to contain pesticidal Cry toxins have been soldto the American farmer. However, while they have proven to be verysuccessful commercially, these genetically engineered, insect-resistantcrop plants provide resistance to only a narrow range of theeconomically important insect pests. Some insects, such as Western cornrootworm, have proven to be recalcitrant, and the level of Bt-toxinresistance is increasing in formerly susceptible populations of someimportant insect pests.

[0008] Although numerous investigators have attempted to make mutantendotoxin proteins with improved insecticidal activity, few havesucceeded. In fact, the majority of genetically engineered B.thuringiensis toxins that have been reported in the literature reportendotoxin activity that is no better than that of the wild-type protein,and in many cases, the activity is decreased or destroyed altogether.Thus, new microbial insecticides having altered specificity and/orimproved pesticidal activity are desired for use in pest-managementstrategies.

SUMMARY OF THE INVENTION

[0009] Compositions and methods are provided for impacting plant pests,particularly Coleopteran insect pests. More specifically, the inventionrelates to methods of impacting insects utilizing nucleic acids derivedfrom δ-endotoxin genes to produce transformed microorganisms and plantsthat express a pesticidal polypeptide of the invention. The compositionsand methods of the invention find use in agriculture for controllingpests of crop plants.

[0010] The invention provides nucleic acids, and fragments and variantsthereof, which encode polypeptides that possess pesticidal activityagainst pests of the order Coleoptera. The wild-type (e.g., naturallyoccurring) nucleotide sequences of the invention, which were obtainedfrom strains of Bacillus thuringiensis, encode Cry-8-like δ-endotoxins.

[0011] The invention further provides fragments and variants of Cry-8like nucleotide sequences that encode biologically active (e.g.,pesticidal) polypeptides. In particular embodiments, the disclosednucleotide sequences encode polypeptides that are pesticidal for atleast one insect belonging to the order Coleopteran (e.g., Coloradopotato beetle, southern corn rootworm, and western corn rootworm).

[0012] Other embodiments of the invention provide nucleic acid encodingtruncated versions of a Cry8 endotoxin that are characterized bypesticidal activity that is either equivalent to, or improved, relativeto the activity of the corresponding full-length endotoxin. Some of thetruncated nucleic acids of the invention can be referred to as eitherfragments or variants. In particular embodiments, some of the nucleicacid fragments/variants of the invention are truncated at the 3′ end ofa wild-type coding sequence; in alternative embodiments, other nucleicacids of the invention comprise a contiguous sequence of nucleic acidresidues, derived from another coding sequence of the invention, thathave been truncated at both the 5′ and 3′ ends.

[0013] The invention also provides recombinant Cry8-like nucleic acidscomprising mutagenized nucleic acid sequence variants encoding B.thuringiensis endotoxins that have been engineered to have improvedand/or altered pesticidal activities. More specifically, the inventionprovides mutagenized nucleic acids encoding pesticidal polypeptides thatcomprise an additional, or an alternative, protease-sensitive sitelocated in domain 1 of the polypeptide variant in a region that islocated between alpha-helices 3 and 4 of the encoded polypeptide.

[0014] As demonstrated herein, the presence of an additional, and/oralternative, protease-sensitive site in the amino acid sequence of theencoded polypeptide can improve the pesticidal activity and/orspecificity of the variant polypeptide encoded by the nucleic acidvariants of the invention. Accordingly, the Cry8-nucleotide sequences ofthe invention can be recombinantly engineered or manipulated to produceendotoxins having improved or altered activity and/or specificitycompared to that of an unmodified wild-type δ-endotoxin.

[0015] For example, one type of variant nucleic acid (e.g., mutagenizedCry8-like nucleotide sequence) disclosed herein provides additionalmutants that comprise additional codons that introduce a secondtrypsin-sensitive amino acid sequence (in addition to the naturallyoccurring trypsin site) into its encoded polypeptide. An alternativeaddition variant of the invention comprises additional codons designedto introduce a chymotrypsin-sensitive site located immediately 5′ of thenaturally occurring trypsin site.

[0016] A second alternative type of variant nucleic acid of theinvention provides substitution mutants in which at least one codon ofthe nucleic acid that encodes the naturally occurring protease-sensitivesite is destroyed, and alternative codons are introduced into thevariant nucleic acid sequence in order to introduce a different (e.g.,substitute) protease-sensitive site in its place. In a particularembodiment of this variant polynucleotide, a replacement mutant isdisclosed in which the naturally occurring trypsin cleavage site presentin the encoded polypeptide is destroyed and a chymotrypsin cleavage siteis introduced into in its place.

[0017] It is to be recognized that any of the disclosed mutations can beengineered in any polynucleotide sequence of the invention thatcomprises the amino acid residues providing the trypsin cleavage sitethat is targeted for modification. Accordingly, variants of eitherfull-length endotoxins or fragments thereof can be modified to containadditional or alternative cleavage sites.

[0018] The nucleic acids of the invention can be used to produceexpression cassettes that can be used to produce transformedmicroorganisms comprising a nucleic acid of the invention. The resultingtransformants can be used in the preparation of pesticidal compositionscomprising a transformed microorganism, or for the production andisolation of pesticidal proteins. Thus, the invention further providespesticidal compositions, comprising either pesticidal polypeptides ortransformed microorganisms, and methods for producing such compositions.The pesticidal compositions of the invention find use in agriculturalmethods for impacting pests. For example, the compositions can be usedin a method that involves placing an effective amount of the pesticidalcomposition in the environment of the pest by a procedure selected fromthe group consisting of spraying, dusting, broadcasting, or seedcoating.

[0019] The invention further provides isolated pesticidal (e.g.,insecticidal) polypeptides encoded by either a naturally occurring, or amodified (e.g., mutagenized or manipulated) nucleic acid of theinvention. In particular examples, pesticidal proteins of the inventioninclude full-length δ-endotoxin proteins, fragments of full-lengthδ-endotoxins, and variant polypeptides that are produced frommutagenized nucleic acids designed to introduce particular amino acidsequences into the polypeptides of the invention. In particularembodiments, the polypeptide fragments and polypeptide variants of theinvention have enhanced pesticidal activity relative to the activity ofthe naturally occurring δ-endotoxin from which they are derived.Polypeptides of the invention can be produced either from a nucleic aciddisclosed herein, or by the use of standard molecular biologytechniques. For example, a truncated protein of the invention can beproduced by expression of a recombinant nucleic acid of the invention inan appropriate host cell, or alternatively by a combination of ex vivoprocedures, such as protease digestion and purification.

[0020] The nucleic acids of the invention can also be used to producetransgenic (e.g., transformed) plants that are characterized by genomesthat comprise at least one stably incorporated nucleotide constructcomprising a coding sequence of the invention operably linked to apromoter that drives expression of the encoded pesticidal polypeptide.Accordingly, transformed plant cells, plant tissues, plants, and seedsthereof are also provided.

[0021] In a particular embodiment, a transformed plant of the inventioncan be produced using a nucleic acid that has been optimized forincreased expression in a host plant. For example, one of the pesticidalpolypeptides of the invention can be back-translated to produced anucleic acid comprising codons optimized for expression in a particularhost, for example a plant, more specifically for expression in a Zeamays plant. Expression of a coding sequence by such a transformed plant(e.g., dicot or monocot) will result in the production of a pesticidalpolypeptide and confer increased insect resistance to the plant. In aparticular embodiment, the invention provides transgenic plantsexpressing pesticidal polypeptides that find use in methods forimpacting the Colorado potato beetle, western corn rootworm, andsouthern corn rootworm.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The invention is drawn to compositions and methods for impactingpests, particularly plant pests, more specifically insect pests of theorder Coleopteran. More specifically, the isolated nucleic acids of theinvention, and fragments and variants thereof, comprise nucleotidesequences that encode pesticidal polypeptides (e.g., proteins). Thedisclosed pesticidal proteins are biologically active (e.g., pesticidal)against insect pests, particularly the Colorado potato beetle(Leptinotarsa decemlineata), the western corn rootworm (Diabroticavirgifera virgifera), and the southern corn rootworm (Diabroticaundecimpunctata howardi).

[0023] The compositions of the invention comprise isolated nucleicacids, and fragments and variants thereof, that encode pesticidalpolypeptides, expression cassettes comprising nucleotide sequences ofthe invention, isolated pesticidal proteins, and pesticidalcompositions. In some embodiments, the invention provides modifiedCry8-like δ-endotoxin proteins characterized by improved insecticidalactivity against Coleopterans relative to the pesticidal activity of thecorresponding wild-type parental protein. The invention further providesplants and microorganisms transformed with these novel nucleic acids,and methods involving the use of such nucleic acids, pesticidalcompositions, and transformed organisms in impacting insect pests.

[0024] In the description that follows, a number of terms are usedextensively. The following definitions are provided to facilitateunderstanding of the invention.

[0025] As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues (e.g., peptide nucleic acids) having the essential nature ofnatural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides.

[0026] As used herein the terms “encoding” or “encoded”, when used inthe context of a specified nucleic acid, means that the nucleic acidcomprises the requisite information to direct translation of thenucleotide sequence into a specified protein. The information by which aprotein is encoded is specified by the use of codons. A nucleic acidencoding a protein may comprise non-translated sequences (e.g., introns)within translated regions of the nucleic acid, or may lack suchintervening non-translated sequences (e.g., as in cDNA).

[0027] As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire nucleicacid sequence or the entire amino acid sequence of, a native(non-synthetic), endogenous sequence. A full-length polynucleotideencodes the full-length, catalytically active form of the specifiedprotein.

[0028] As used herein, the term “antisense” used in the context oforientation of a nucleotide sequence refers to a duplex polynucleotidesequence that is operably linked to a promoter in an orientation wherethe antisense strand is transcribed. The antisense strand issufficiently complementary to an endogenous transcription product suchthat translation of the endogenous transcription product is ofteninhibited.

[0029] The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

[0030] The terms “residue” or “amino acid residue” or “amino acid” areused interchangeably herein to refer to an amino acid that isincorporated into a protein, polypeptide, or peptide (collectively“protein”). The amino acid may be a naturally occurring amino acid and,unless otherwise limited, may encompass known analogues of natural aminoacids that can function in a similar manner as naturally occurring aminoacids.

[0031] As used herein the terms “isolated” and “purified” are usedinterchangeably to refer to nucleic acids, or polypeptides, orbiologically active portion thereof, that are substantially oressentially free from components that normally accompany or interactwith the nucleic acid or polypeptide as found in its naturally occurringenvironment. Thus, an isolated or purified nucleic acid or polypeptideis substantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

[0032] As used herein the term “impacting insect pests” refers toeffecting changes in insect feeding, growth, and/or behavior at anystage of development, including, but not limited to, killing the insect,retarding growth, preventing reproductive capability, and the like.

[0033] As used herein the terms “pesticidal activity” and “insecticidalactivity” are used synonymously to refer to activity of an organism or asubstance, such as, for example, a protein, that can be measured by, butis not limited to, pest mortality, pest weight loss, pest attraction,pest repellency, and other behavioral and physical changes of a pestafter feeding and exposure for an appropriate length of time. Forexample “pesticidal proteins” are proteins that display pesticidalactivity by themselves or in combination with other proteins.

[0034] The term “pesticidally effective amount” connotes a quantity of asubstance or organism that has pesticidal activity when present in theenvironment of a pest. For each substance or organism, the pesticidallyeffective amount is determined empirically for each pest affected in aspecific environment. Similarly an “insecticidally effective amount” maybe used to refer to an “pesticidally effective amount” when the pest isan insect pest.

[0035] As used herein the term “recombinantly engineered” connotes theutilization of recombinant DNA technology to introduce (e.g., engineer)a change in the protein structure based on an understanding of theprotein's mechanism of action and a consideration of the amino acidsbeing introduced, deleted or substituted.

[0036] As used herein the term “mutagenized nucleotide sequence”connotes a nucleotide sequence that has been mutagenized or altered tocontain one or more nucleotide residues (e.g., base pair) that is notpresent in the corresponding wild-type sequence, and which encodes amutant δ-endotoxin showing improved insecticidal activity.

[0037] As used herein the term “improved insecticidal activity”characterizes a δ-endotoxin of the invention that either has enhancedanti-Coleopteran pesticidal activity relative to the activity of itscorresponding wild-type protein, and/or an endotoxin that is effectiveagainst either a broader range of insects, or acquires a specificity foran insect that is not susceptible to the toxicity of the wild-typeprotein. A finding of enhanced pesticidal activity requires ademonstration of an increase of toxicity of at least 30% against theinsect target, and more preferably 35%, 40%, 45%, or 50% relative to theinsecticidal activity of the wild-type endotoxin determined against thesame insect.

[0038] Units, prefixes, and symbols may be denoted in their SI acceptedform. Unless otherwise indicated, nucleic acids are written left toright in 5′ to 3′ orientation; amino acid sequences are written left toright in amino to carboxy orientation, respectively. Numeric ranges areinclusive of the numbers defining the range. Amino acids may be referredto herein by either their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The above-defined terms are more fullydefined by reference to the specification as a whole.

[0039] The nucleotide sequences of the invention may be used totransform any organism to produce the encoded pesticidal proteins.Methods are provided that involve the use of such transformed organismsto impact or control plant pests. The invention further relates to theidentification of fragments and variants of the naturally occurringcoding sequence that encode biologically active pesticidal proteins. Allof the nucleotide sequences of the invention find direct use in methodsfor impacting pests, particularly insect pests, more particularly pestsof the order Coleopteran, including, for example, the Colorado potatobeetle, western corn rootworm, and southern corn rootworm. Accordingly,the present invention provides new approaches for impacting insect peststhat do not depend on the use of traditional, synthetic chemicalinsecticides. The invention involves the discovery of naturallyoccurring, biodegradable pesticides and the genes that encode them.

[0040] The invention further provides fragments and variants of thenaturally occurring coding sequences that also encode biologicallyactive (e.g., pesticidal) polypeptides. The nucleic acids of theinvention encompass nucleic acid sequences that have been optimized forexpression by the cells of a particular organism, for example nucleicacid sequences that have been back-translated using plant-preferredcodons based on the amino acid sequence of a polypeptide having enhancedpesticidal activity.

[0041] The nucleotide sequences of the invention were isolated fromstrains of the bacterium, Bacillus thuringiensis. Crude lysates preparedfrom cultures of the strains were discovered to have pesticidal activityagainst Colorado potato beetle, western corn rootworm, and southern cornrootworm. Crystal proteins were isolated from cultures of the strains.The isolated crystal proteins were tested for pesticidal activity ininsect feeding assays. The results of the assays revealed that theisolated crystal proteins possessed Coleopteran pesticidal activity. Aneffort was undertaken to identify nucleotide sequences encoding crystalproteins from the strains, and the naturally occurring coding sequencesand genomic nucleic acids of the invention were discovered.

[0042] The nucleotide sequences of the isolated nucleic acids weredemonstrated to encode pesticidal proteins by transforming Escherichiacoli with such nucleotide sequences. Lysates prepared from thetransformed E. coli had pesticidal activity against corn rootworms andColorado potato beetles in feeding assays, demonstrating that theisolated nucleotide sequences of the invention encode pesticidalproteins. Depending upon the characteristics of a given lysatepreparation, it was recognized that the demonstration of pesticidalactivity sometimes required trypsin pretreatment to activate thepesticidal proteins.

[0043] Subsequently, nucleic acid variants and fragments encodingbiologically active pesticidal polypeptides were identified. Some of theencoded pesticidal proteins require protease (e.g., trypsin) activationand other proteins were observed to be biologically active (e.g.,pesticidal) in the absence of activation. In some embodiments, thenucleic acid encodes a truncated version of the naturally occurringpolypeptide and as such, can be classified either as a variant or afragment. In addition, second generation nucleic acid sequences wereengineered to comprise nucleotide sequences that encode Cry8-likepolypeptides characterized by improved or altered pesticidal activityrelative to the pesticidal activity of the naturally occurringpolypeptide.

[0044] The nucleic acids of the invention comprise isolatedpolynucleotides, and variants and fragments thereof, that encodebiologically active (e.g., pesticidal) polypeptides, including, but notlimited to, the Cry8-like nucleotide sequences set forth in SEQ IDNOS:1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 27, 28, 29, 31, 33, 39, 41,43, and 45. The nucleotide sequences disclosed herein provide twobackground sequences referred to herein as 1218-1 and 49PVD into whichmutations are introduced. In some instances, the sequences also providevariants of two distinct clones referred to herein as 1218-1 and 1218-2.More specifically, SEQ ID NO:15 (1218-1A) represents a variant of SEQ IDNO:5, each of which represent alternative embodiments of the 1218-1clone. In addition, SEQ ID NO:17 (1218-2A) represents a variant of SEQID NO:7; each of which represent alternative embodiments of the 1218-2clone.

[0045] The polynucleotides of the invention also include any syntheticor recombinant nucleotide sequence that encodes a pesticidal polypeptidecomprising the amino acid sequences set forth in SEQ ID NOS: 2, 4, 6, 8,10, 12, 16, 18, 20, 22, 24, 30, 32, 34, 40, 42, 44, and 46.

[0046] An “isolated” nucleic acid is free of sequences (preferablyprotein encoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acids can containless than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb ofnucleotide sequences that naturally flank the nucleic acids in genomicDNA of the cell from which the nucleic acid is derived.

[0047] The present invention provides isolated nucleic acids comprisingnucleotide sequences which encode the amino acid sequences set forth inSEQ ID NOS: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 30, 32, 34, 40, 42,44, and 46. In particular embodiments, the invention provides nucleicacids comprising the nucleotide sequences set forth in SEQ IDNOS:1(Cry1218-1 CDS) and 3 (Cry1218-2 CDS), the maize-optimized nucleicacid set forth in SEQ ID NO:9 (mo1218-1), and the native genomicsequences set forth in SEQ ID NO:27 (genomic Cry1218-1) and SEQ ID NO:28(genomic Cry 1218-2). The coding sequence (CDS) for SEQ ID NO: 27 runsfrom base pair 731-4348. The CDS for SEQ ID NO: 28 runs from base pair1254-4883. Plasmids comprising each of these five nucleic acids weredeposited on May 5, 2000 and Nov. 2, 2000 with the Patent Depository ofthe American Type Culture Collection (ATCC), Manassas, Va., and assignedPatent Deposit Nos. PTA-1821 (corresponding to SEQ ID NO:1); PTA-1817(corresponding to SEQ ID NO:3); PTA-2635 (corresponding to SEQ ID NO:9);PTA-2634 (comprising SEQ ID NO:27); and PTA-2636 (comprising SEQ IDNO:28).

[0048] Patent Deposits PTA-1821 and PTA-1817 comprise a mixture of 2clones, each of which contains a part of the entire coding sequence.More specifically, the deposited plasmids encode nucleic acid moleculescloned into a TA vector (Invitrogen, Carlsbad, Calif.) that encode twooverlapping fragments of the coding sequence. The full length codingsequence can be produced using an overlapping PCR strategy. A first PCRreaction should comprise forward and reverse primers designed tocorrespond to the 5′ and the 3′ ends of the full-length coding sequence.Suitable primers for use in PCR reactions are set forth in SEQ ID NOS:35through 38. More specifically, SEQ ID NOS:35 and 36 provide a firstprimer set “(a)” comprising a forward primer SEQ ID NO:35(5′-ATGAGTCCAAATAATCAAAATG) and a reverse primer SEQ ID NO:36(5′-CCGCTTCTAAATCTTGTTCC) for the 5′ end of the coding sequence. SEQ IDNOS:37 and 38 provide a second primer set “(b)” comprising a forwardprimer SEQ ID NO:37 (5′-GGAACAAGATTTAGAGG) and a reverse primer SEQ IDNO:38 (5′-CTCATCGTCTACAATCAATTCATC) for the 3′ end of the codingsequence. The two DNA bands generated by the first PCR reactionperformed with the above-identified primer sets should be purified and asecond round of PCR, set for 7 cycles, should be performed utilizing thepurified DNA isolated from the first PCR reaction in the absence of anyprimers. The 3′ end of the nucleic acid generated by primer set (a) andthe 5′ end of the nucleic acid generated by primer set (b) will overlapand prime the generation of the full-length coding sequence. A third andfinal PCR reaction is performed to generate the full-length codingsequence. This reaction is performed using 1 μl of the second PCRreaction product and a primer set comprising SEQ ID NO:35 (forwardprimer of set (a)) and SEQ ID NO:39 (reverse primer of set (b)).

[0049] The above-referenced deposits (e.g., PTA-1821; PTA-1817;PTA-2635; PTA-2634; and PTA-2636) will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. These deposits weremade merely as a convenience for those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112.

[0050] Of particular interest are optimized nucleotide sequencesencoding the pesticidal proteins of the invention. As used herein thephrase “optimized nucleotide sequences” refers to nucleic acids that areoptimized for expression in a particular organism, for example a plant.Optimized nucleotide sequences may be prepared for any organism ofinterest using methods known in the art. For example, SEQ ID NO:9discloses an optimized nucleic acid sequence encoding the pesticidalprotein set forth in SEQ ID NO:16 (truncated 1218-1A). Morespecifically, the nucleotide sequence of SEQ ID NO:9 comprisingmaize-preferred codons SEQ ID NO:9 was prepared by reverse-translatingthe amino acid sequence set forth in SEQ ID NO:16 to comprisemaize-preferred codons as described by Murray et al. (1989) NucleicAcids Res. 17:477-498. Optimized nucleotide sequence find use inincreasing expression of a pesticidal protein in a plant, particularly amonocot plant, more particularly a plant of the Gramineae (Poaceae)family, most particularly a maize or corn plant.

[0051] The invention further provides isolated pesticidal (e.g.,insecticidal) polypeptides encoded by either a naturally occurring, or amodified (e.g., mutagenized or truncated) nucleic acid of the invention.More specifically, the invention provides polypeptides comprising anamino acid sequence set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18,20, 22, 24, 30, 32, 34, 40, 42, 44, and 46 and the polypeptides encodedby a nucleic acids described herein, for example those set forth in SEQID NOS:1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 27, 28, 29, 31, 33, 39,41, 43, and 45, and fragments and variants thereof.

[0052] In particular embodiments, pesticidal proteins of the inventionprovide full-length δ-endotoxin proteins, fragments of full-lengthδ-endotoxins, and variant polypeptides that are produced frommutagenized nucleic acids designed to introduce particular amino acidsequences into polypeptides of the invention. In particular embodiments,the amino acid sequences that are introduced into the polypeptidescomprise a sequence that provides a cleavage site for an enzyme orprotease.

[0053] Some of the polypeptides of the invention, for example SEQ IDNOS:2 and 4 comprise full-length δ-endotoxins; other polypeptides suchas SEQ ID NOS:6, 8, 10, 16, 18, and 20 embody fragments of a fill-lengthδ-endotoxin; and SEQ ID NOS:12, 22, 24, 30, 32, 34, 40, 42, 44, and 46provide polypeptide variants. Some of the polypeptide fragments andvariants of the invention have enhanced pesticidal activity relative tothe activity of the naturally occurring δ-endotoxin from which they arederived, particularly in the absence of in vitro activation of theendotoxin with a protease prior to screening for activity. For example,the data presented herein in Table 1 of Example 6 indicates that theNGRS addition mutant (SEQ ID NO:12) of SEQ ID NO:16 (truncated 1218-1Aendotoxin) is characterized by increased pesticidal activity againstColorado potato beetle.

[0054] SEQ ID NOS:6, 10, 16 and 20 provide polypeptides that embodytruncated versions of the 1218-1 polypeptide set forth in SEQ ID NO:2.SEQ ID NO:16 provides a variant, referred to herein as 1218-1A of thepolypeptide set forth in SEQ ID NO:6 and referred to herein as 1218-1.Three of the above-mentioned sequences, SEQ ID NOS: 6, 10 and 16represent a polypeptide that is shortened (truncated) at the 3′ end ofthe amino acid sequence set forth in SEQ ID NO:2. In contrast, thefourth polypeptide variant set forth in SEQ ID NO:20 provides a variantthat is truncated at both the 5′ and 3′ ends of the full-length proteinset forth in SEQ ID NO:2. SEQ ID NOS: 8 and 18 (1218-2 and 1218-2A,respectively) provide polypeptides that embody truncated versions of thepolypeptides set forth in SEQ ID NO:4. Each of these two polypeptidesprovide a protein that is truncated at the 3′ end of the full-length1218-2 polypeptide set forth in SEQ ID NO:4.

[0055] SEQ ID NOS: 12, 22, 24, 40, and 44 provide a family ofpolypeptides that embody variants of the 1218-1A truncated polypeptidesset forth in SEQ ID NO:16, thus SEQ ID NOS:12, 22, 24, 40, and 44provide variants (or mutants) of the biologically active fragment of theCry8-like polypeptide set forth in SEQ ID NO:2. More specifically, SEQID NO:12 provides a mutant, referred to herein as NGSR.N1218-1, thatcomprises an additional trypsin-sensitive cleavage site; SEQ ID NO:22provides a second mutant, referred to herein as LKMS.N1218-1, thatcomprises a chymotrypsin-sensitive cleavage site that is not present inthe wild-type 1218-1 or 1218-1A polypeptide; and SEQ ID NO:24 provides areplacement mutant, referred to herein as LKMS.R1218-1, in which anexisting trypsin cleavage-site is destroyed and a chymotrypsin site isintroduced in its place. SEQ ID NO:40 provides a secondchymotrypsin-addition mutant, referred to herein as LRMS.N1218-1, thatcomprises the alternative chymotrypsin cleavage site LRMS (SEQ IDNO:48). SEQ ID NO:44 provides a second replacement or substitutionmutant, referred to herein as LRMS.R1218-1, in which the native trypsinsite is replaced with the chymotrypsin cleavage site LRMS.

[0056] SEQ ID NOS:30, 32, 34, 42, and 46 provide a second family ofpolypeptides that embody variants or mutants of the truncatedpolypeptide set forth in SEQ ID NO:20. Thus, SEQ ID NOS: 30, 32, 34, 42,and 46 provide variants of the pesticidal fragment of SEQ ID NO: 2 thatis set forth in SEQ ID NO: 20. More specifically, SEQ ID NO:30 providesa mutant, referred to herein as NGSR.N49PVD, that comprises anadditional trypsin-senstive cleavage site; SEQ ID NO: 32 provides asecond mutant, referred to herein as LKMS.N49PVD, that comprises achymotrypsin-sensitive cleavage site that is not present in thewild-type 1218-1 or 1218-1A polypeptide; and SEQ ID NO: 34 provides areplacement mutant, referred to herein as LKMS.R49PVD, in which anexisting trypsin cleavage site is destroyed and a chymotrypsin site isintroduced in its place. SEQ ID NO:42 provides a second chymotrypsinaddition mutant, referred to herein as LRMS.N49PVD, that comprises thealternative chymotrypsin cleavage site LRMS (SEQ ID NO:48). SEQ ID NO:46(LRMS.R49PVD) provides a second replacement or substitution mutant inwhich the native trypsin site is replaced with the chymotrypsin cleavagesite LRMS.

[0057] It is to be understood that the polypeptides of the invention canbe produced either by expression of a nucleic acid disclosed herein, orby the use of standard molecular biology techniques. For example, atruncated protein of the invention can be produced by expression of arecombinant nucleic acid of the invention in an appropriate host cell,or alternatively by a combination of ex vivo procedures, such asprotease digestion and purification of a purified wild-type protein.

[0058] As used herein the term “isolated” or “purified” as it is used torefer to a polypeptide of the invention means that the isolated proteinis substantially free of cellular material and includes preparations ofprotein having less than about 30%, 20%, 10%, or 5% (by dry weight) ofcontaminating protein. When the protein of the invention or biologicallyactive portion thereof is recombinantly produced, preferably culturemedium represents less than about 30%, 20%, 10%, or 5% (by dry weight)of chemical precursors or non-protein-of-interest chemicals.

[0059] It is recognized that the pesticidal proteins may be oligomericand will vary in molecular weight, number of residues, componentpeptides, activity against particular pests, and in othercharacteristics. However, by the methods set forth herein, proteinsactive against a variety of pests may be isolated and characterized. Thepesticidal proteins of the invention can be used in combination with Btendotoxins or other insecticidal proteins to increase insect targetrange. Furthermore, the use of the pesticidal proteins of the presentinvention in combination with Bt δ-endotoxins or other insecticidalprinciples of a distinct nature has particular utility for theprevention and/or management of insect resistance. Other insecticidalprinciples include protease inhibitors (both serine and cysteine types),lectins, α-amylase, and peroxidase.

[0060] Fragments and variants of the nucleotide and amino acid sequencesand the polypeptides encoded thereby are also encompassed by the presentinvention. As used herein the term “fragment” refers to a portion of anucleotide sequence of a polynucleotide or a portion of an amino acidsequence of a polypeptide of the invention. Fragments of a nucleotidesequence may encode protein fragments that retain the biologicalactivity of the native protein and hence possess pesticidal activity.Thus, it is acknowledged that some of the polynucleotide and amino acidsequences of the invention can correctly be referred to as eitherfragments and variants. This is particularly true of truncated sequencesthat are biologically active.

[0061] It is to be understood that the term “fragment,” as it is used torefer to nucleic acid sequences of the invention, also encompassessequences that are useful as hybridization probes. This class ofnucleotide sequences generally do not encode fragment proteins retainingbiological activity. Thus, fragments of a nucleotide sequence may rangefrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides, and up to the full-length nucleotide sequence encoding theproteins of the invention.

[0062] A fragment of a Cry8-like nucleotide sequence that encodes abiologically active portion of a pesticidal protein of the inventionwill encode at least 15, 25, 30, 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1,000, 1,100, or 1,200 contiguous amino acids, or up to thetotal number of amino acids present in a pesticidal polypeptide of theinvention (for example, 1,206, 1,210, 667, 667, and 669 amino acids forSEQ ID NOS:2, 4, 6, 8, and 10, respectively). Fragments of a Cry8-likenucleotide sequence that are useful as hybridization probes or PCRprimers generally need not encode a biologically active portion of apesticidal protein.

[0063] Thus, a fragment of a Cry8-like nucleic acid may encode abiologically active portion of a pesticidal protein, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of a pesticidalprotein can be prepared by isolating a portion of one of the Cry8-likenucleotide sequences of the invention, expressing the encoded portion ofthe pesticidal protein (e.g., by recombinant expression in vitro), andassessing the activity of the encoded portion of the pesticidal protein.

[0064] Nucleic acids that are fragments of a Cry8-like nucleotidesequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 1,000, 1,200, 1,400, 1,600, 1,800, 2,000,2,200, 2,400, 2,600, 2,800, 3,000, 3,200, 3,400, or 3,600 nucleotides,or up to the number of nucleotides present in a Cry8-like nucleotidesequence disclosed herein (for example, 3,621, 3,633, 2,003, 2,003,2,010, and 2010 and 2022 nucleotides for SEQ ID NOS:1, 3, 5, 7, 9, 15and 17 respectively).

[0065] For example, SEQ ID NOS: 5, 9, 15, and 19 represent fragments ofSEQ ID NO:1 and SEQ ID NOS:7 and 17 represent fragments of SEQ ID NO: 3.More specifically, particular embodiments of the nucleic acids of theinvention disclose fragments derived from (e.g., produced from) a firstnucleic acid of the invention, wherein the fragment encodes a truncatedCry8-like endotoxin characterized by pesticidal activity. The truncatedpolypeptide encoded by the polynucleotide fragments of the invention arecharacterized by pesticidal activity that is either equivalent to, orimproved, relative to the activity of the corresponding full-lengthpolypeptide encoded by the first nucleic acid from which the fragment isderived.

[0066] In specific embodiments, some of the nucleic acid fragments ofthe invention are truncated at the 3′ end of the wild-type codingsequence. For example, SEQ ID NOS: 5 and 15 represent fragments of SEQID NO: 1 that are truncated at the 3′ end. In an alternative embodiment,one of the polynucleotides of the invention, SEQ ID NO: 19, comprises anucleic acid sequence that is truncated at both the 5′ and 3′ end of thetruncated 1218-1 and 1218-1A toxin domain encoded by SEQ ID NOS: 5 and15, respectively.

[0067] By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the pesticidal polypeptides of the invention.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below.

[0068] Variant nucleotide sequences also include synthetically derivednucleotide sequences, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a pesticidal protein ofthe invention. Generally, variants of a particular nucleotide sequenceof the invention will have at least about 40%, 50%, 60%, 65%, 70%,generally at least about 75%, 80%, 85%, preferably at least about 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably atleast about 98%, 99%, or more sequence identity to that particularnucleotide sequence as determined by sequence alignment programsdescribed elsewhere herein using default parameters.

[0069] As used herein the term “variant protein” encompassespolypeptides that are derived from a native protein by deletion(so-called truncation) or addition of one or more amino acids to theN-terminal and/or C-terminal end of the native protein; deletion oraddition of one or more amino acids at one or more sites in the nativeprotein; or substitution of one or more amino acids at one or more sitesin the native protein. Accordingly, the term variant protein encompassesbiologically active fragments of a native protein that comprise asufficient number of contiguous amino acid residues to retain thebiological activity of the native protein.

[0070] Variant proteins encompassed by the present invention arebiologically active, that is they continue to possess the desiredbiological activity of the native protein, that is, pesticidal activityas described herein. Such variants may result from, for example, geneticpolymorphism or from human manipulation. Biologically active variants ofa native pesticidal protein of the invention will have at least about40%, 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%,preferably at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, and more preferably at least about 98%, 99%, or moresequence identity to the amino acid sequence for the native protein asdetermined by sequence alignment programs described elsewhere hereinusing default parameters. A biologically active variant of a protein ofthe invention may differ from that protein by as few as 1-15 amino acidresidues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2,or even 1 amino acid residue.

[0071] It is recognized that the nucleic acid sequence of any one of thepolynucleotides of the invention can be altered or mutagenized to alter(e.g., improve) the biological activity and/or specificity of itsencoded pesticidal polypeptide. For example, SEQ ID NO: 11 represents aCry8-like nucleotide sequence that has been mutagenized to comprise 12additional nucleotides (SEQ ID NO:13) that are not present in thewild-type nucleic acid sequence (SEQ ID NO: 15) that is being altered.The nucleotide sequence inserted into the coding region of SEQ ID NO: 15was designed to encode an NGRS addition mutant that comprises anadditional trypsin cleavage site (NGSR) (SEQ ID NO:14) in the amino acidsequence of the encoded polypeptide.

[0072] More specifically, the amino acid sequence set forth in SEQ IDNO:14 was introduced between amino acid 164 and 165 of the Cry8δ-endotoxin set forth in SEQ ID NO:16. This particular amino acidsequence was chosen because it duplicates the endogenous sequencepresent in the naturally occurring full-length protein (SEQ ID NO:2),and creates a second protease-sensitive site. More specifically, themodification introduces a second trypsin-like site. It is well known tothose of skill in the art that trypsin cleaves bonds immediatelyC-terminal to arginine and lysine. As demonstrated herein therecombinantly engineered protein (SEQ ID NO:12) encoded by SEQ ID NO:11is characterized by improved activity against Coleopterans, particularlyagainst Colorado potato beetle (see Example 6, Table 1), southern cornrootworm (see Example 7, Tables 2 through 4 and 6), and western cornrootworm (see Example 7, Table 5).

[0073] SEQ ID NO: 21 represents a Cry8-like nucleotide sequence that hasbeen mutagenized to comprise 12 additional nucleotides (SEQ ID NO:25)that are not present in the wild-type endotoxin. The inserted nucleotidesequence was designed to encode an LKMS addition mutant that comprises achymotrypsin cleavage site (LKMS) (SEQ ID NO:26) in the amino acidsequence of the encoded polypeptide. More specifically, the LKMSaddition mutant (LKMS.N1218-1) comprises a nucleotide sequence insertthat introduces the amino acid sequence LKMS between amino acids 160 and161 of SEQ ID NO:6. The LKMS replacement mutant LKMS.R1218-1 comprises apolypeptide in which the amino acid sequence LKMS is introduced betweenamino acid 160 and 161 of SEQ ID NO:16 and the amino acids NGS areremoved from amino acid positions 161-163 of SEQ ID NO:16. Thismodification removes a trypsin site and introduces a chymotrypsin site.Chymotrypsin cleaves bonds immediately C-terminal to Methionine.

[0074] The LRMS addition mutant (LRMS.N1218-1) and replacement mutant(LRMS.R1218-1) provide alternative embodiments of polypeptidescomprising an additional or alternative chymotrypsin cleavage site, butthe LRMS mutants differ in the specific amino acid sequence (SEQ ID NO:48) and nucleotide sequence (SEQ ID NO: 47) that is used to introducethe chymotrypsin cleavage site into the nucleic acid sequence thatencodes the mutant polypeptides.

[0075] SEQ ID NO: 30 (NGSR.N49PVD), SEQ ID NO: 32 (LKMS.N49PVD), SEQ IDNO: 34 (LKMS.R49PVD), SEQ ID NO: 42 (LRMS.N49PVD), and SEQ ID NO: 46(LRMS.R49PVD) provide mutants of the truncated pesticidal polypeptide49PVD. The amino acid sequence of 49PVD is provided in SEQ ID NO: 20.The basic design of the these polypeptides and their nomenclature followthe same pattern discussed above for the 1218-1 truncated polypeptide,and are explained more fully elsewhere herein.

[0076] It is recognized that any nucleotide sequence encoding the aminoacid sequences NGSR, LKMS, or LRMS can be used and that the exactidentity of the codons used to introduce any of these cleavage sitesinto a variant polypeptide may vary depending on the use, i.e.,expression in particular plant species. It is also recognized that anyof the disclosed mutations can be introduced into any polynucleotidesequence of the invention that comprises the codons for amino acidresidues that provide the native trypsin cleavage site that is targetedfor modification. Accordingly, variants of either full-length endotoxinsor fragments thereof can be modified to contain additional oralternative cleavage sites, and these embodiments are intended to beencompassed by the scope of the invention disclosed and claimed herein.

[0077] The invention further encompasses a microorganism that istransformed with at least one nucleic acid of the invention, with anexpression cassette comprising the nucleic acid, or with a vectorcomprising the expression cassette. Preferably, the microorganism is onethat multiplies on plants. More preferably, the microorganism is aroot-colonizing bacterium. An embodiment of the invention relates to anencapsulated pesticidal protein, which comprises a transformedmicroorganism comprising at least one pesticidal protein of theinvention.

[0078] The invention provides pesticidal compositions comprising atransformed organism of the invention. Preferably the transformedmicroorganism is present in the pesticidal composition in a pesticidallyeffective amount, together with a suitable carrier. The invention alsoencompasses pesticidal compositions comprising an isolated protein ofthe invention, alone or in combination with a transformed organism ofthe invention and/or an encapsulated pesticidal protein of theinvention, in an insecticidally effective amount, together with asuitable carrier.

[0079] The invention further provides a method of increasing insecttarget range by using a pesticidal protein of the invention incombination with at least one second pesticidal protein that isdifferent from the pesticidal protein of the invention. Any pesticidalprotein known in the art can be employed in the methods of the presentinvention. Such pesticidal proteins include, but are not limited to, Btδ-endotoxins, protease inhibitors, lectins, α-amylases, and peroxidases.

[0080] The invention also encompasses transformed or transgenic plantscomprising at least one nucleotide sequence of the invention.Preferably, the plant is stably transformed with a nucleotide constructcomprising at least one nucleotide sequence of the invention operablylinked to a promoter that drives expression in a plant cell. As usedherein, the terms “transformed plant” and “transgenic plant” refer to aplant that comprises within its genome a heterologous polynucleotide.Generally, the heterologous polynucleotide is stably integrated withinthe genome of a transgenic or transformed plant such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant expression cassette.

[0081] It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant, thegenotype of which has been altered by the presence of heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

[0082] As used herein, the term “plant” includes reference to wholeplants, plant organs (e.g., leaves, stems, roots, etc.), seeds, andplant cells, and progeny of same. Parts of transgenic plants are to beunderstood within the scope of the invention to comprise, for example,plant cells, protoplasts, tissues, callus, embryos as well as flowers,stems, fruits, leaves, roots originating in transgenic plants or theirprogeny previously transformed with a DNA molecule of the invention andtherefore consisting at least in part of transgenic cells, are also anobject of the present invention.

[0083] As used herein the term “plant cell” includes, withoutlimitation, seeds suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores. The class of plants that can be used in the methods ofthe invention is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants. A preferred plant is Solanum tuberosum. Aparticularly preferred plant is Zea mays.

[0084] While the invention does not depend on a particular biologicalmechanism for increasing the resistance of a plant to a plant pest,expression of the nucleotide sequences of the invention in a plant canresult in the production of the pesticidal proteins of the invention andin an increase in the resistance of the plant to a plant pest. Theplants of the invention find use in agriculture in methods for impactinginsect pests. Certain embodiments of the invention provide transformedmaize plants, which find use in methods for impacting western andsouthern corn rootworms. Another embodiment of the invention providestransformed potato plants, which find use in methods for impacting theColorado potato beetle.

[0085] One of skill in the art will readily acknowledge that advances inthe field of molecular biology such as site-specific and randommutagenesis, polymerase chain reaction methodologies, and proteinengineering techniques provide an extensive collection of tools andprotocols suitable for use to alter or engineer both the amino acidsequence and underlying genetic sequences, of proteins of agriculturalinterest. Thus, the Cry8-like proteins of the invention may be alteredin various ways including amino acid substitutions, deletions,truncations, and insertions. Methods for such manipulations aregenerally known in the art.

[0086] For example, amino acid sequence variants of the pesticidalproteins can be prepared by introducing mutations into a syntheticnucleic acid (e.g, DNA molecule). Methods for mutagenesis and nucleicacid alterations are well known in the art. For example, designedchanges can be introduced using an oligonucleotide-mediatedsite-directed mutagenesis technique. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York), and the references cited therein.

[0087] The wild-type (e.g., naturally occurring) nucleotide sequences ofthe invention were obtained from strains of Bacillus thuringiensisencoding Cry8-like δ-endotoxins. It is well known that naturallyoccurring δ-endotoxins are synthesized by B. thuringiensis sporulatingcells as a proteinaceous crystalline inclusion protoxin. Upon beingingested by susceptible insect larvae, the microcrystals dissolve in themidgut, and the protoxin is transformed into a biologically activemoiety by proteases characteristic of digestive enzymes located in theinsect gut. The activated δ-endotoxin binds with high affinity toprotein receptors on brush-border membrane vesicles. The epithelialcells lining the midgut are the primary target of the endotoxin and arerapidly destroyed as a consequence of membrane perforation resultingfrom the formation of gated, cation-selective channels by the toxin.

[0088] A comparison of the amino acid sequences of Cry toxins ofdifferent specificities reveals five highly conserved sequence blocks.Structurally, the δ-endotoxins comprise three distinct domains, whichare, from the N- to C-termini: a cluster of seven alpha-helicesimplicated in pore formation, three anti-parallel beta sheets implicatedin cell binding, and a beta sandwich.

[0089] The mutant Cry8 polypeptides of the present invention weregenerally prepared by a process that involved the steps of: obtaining anucleic acid sequence encoding a Cry8 polypeptide; analyzing thestructure of the polypeptide to identify particular “target” sites formutagenesis of the underlying gene sequence, based on a consideration ofthe proposed function of the target domain in the mode of action of theendotoxin; introducing one or more mutations into the nucleic acidsequence to produce a desired change in one or more amino acid residuesof the encoded polypeptide sequence, wherein the change is designed toadd a protease-sensitive cleavage site to the target region or to removethe original protease-sensitive site and to add a protease-sensitivesite that is sensitive to the activity of a different protease; andexpressing the mutagenized nucleic acid sequence that encodes therecombinantly engineered protein of the invention in a transformed hostcell under conditions effective to obtain expression of the modifiedCry8 polypeptide.

[0090] Many of the δ-endotoxins are related to various degrees bysimilarities in their amino acid sequences and tertiary structure, andmeans for obtaining the crystal structures of B. thuringiensisendotoxins are well known. Exemplary high-resolution crystal structuresolution of both the Cry3A and Cry3B polypeptides are available in theliterature. The inventors of the present invention used the solvedstructure of the Cry3A gene (Li et al. (1991) Nature 353:815-821) toproduce a homology model of the Cry8 δ-endotoxin disclosed and claimedherein as SEQ ID NO:2 to gain insight into the relationship betweenstructure and function of the endotoxin, and to design the recombinantlyengineered proteins disclosed and claimed herein. A combinedconsideration of the published structural analyses of B. thuringiensisendotoxins and the reported function associated with particularstructures, motifs, and the like indicates that specific regions of theendotoxin are correlated with particular functions and discrete steps ofthe mode of action of the protein. For example, δ-endotoxins isolatedfrom B. thuringiensis are generally described as comprising threedomains, a seven-helix bundle that is involved in pore formation, athree-sheet domain that has been implicated in receptor binding, and abeta-sandwich motif (Li et al. (1991) Nature, 305:815-821).

[0091] The inventors reasoned that the toxicity of Cry8-like proteins,and specifically the toxicity of the Cry8 protein, could be improved bytargeting the region located between alpha helices 3 and 4 of domain 1of the endotoxin protein. This theory was premised both on the knowledgethat alpha helices 4 and 5 of domain 1 of Cry3A δ-endotoxins had beenreported to insert into the lipid bilayer of cells lining the midgut ofsusceptible insects (Gazit et al., (1998) PNAS USA 95:12289-12294); theinventors' knowledge of the location of trypsin and chymotrypsincleavage cites within the amino acid sequence of the wild-type protein;and the observation reported herein that the protein encoded by 1218-1(i.e., SEQ ID NO:2) was more active against certain Coleopteransfollowing in vitro activation by trypsin or chymotrypsin treatment.Accordingly, the inventors engineered a mutant Cry8-like protein thatwould comprise at least one additional trypsin cleavage site in theregion located between helices 3 and 4 of domain 1.

[0092] More specifically, the inventors produced mutagenized Cry8-likenucleotide sequences that encode mutant Cry8 endotoxins (e.g.,polypeptides) that comprise either additional, or alternativeprotease-sensitive sites. The invention provides mutant polypeptidesthat have been constructed in either a 1218-1 (SEQ ID NOS:6 or 16), or a49PVD (SEQ ID NO:20) background. It should be understood that thedesignation 1218-1 as used herein encompasses two embodiments (e.g.,1218-1 and 1218-1A) of the 1218-1 nucleotide and amino acid sequencespresented herein. This is particularly true in the context of thedisclosed addition and replacement mutants that have been created ineither the 1218-1 or 49PVD background. It is to be understood that thenomenclature used herein to refer to a mutant such as, for example theNGSR.N1218-1 mutant described contemplates mutants created in either the1218-1 and the 1218-1A background. For the sake of consistency, thesequences presented in the sequence listing for the 1218-1 mutantsembody mutants created in the 1218-1A sequences (SEQ ID NOS:15 and 16).

[0093] Generally speaking, all of the mutant polypeptides describedherein are designed to comprise at least one proteolytic cleavage sitelocated between helix 3 and 4 of domain 1 that is not present in thewild-type polypeptide. All of the mutants disclosed herein were clonedinto the pET expression system, expressed in E. coli, and tested forpesticidal activity first against southern corn rootworm (SCRW) and thenwestern corn rootworm (WCRW). Additionally, the 49PVD variant (SEQ IDNO:20) and the NGSR.N1218-1 mutant (SEQ ID NO:12) were tested forpesticidal activity against the Colorado potato beetle (CPB).

[0094] Briefly, the mutants provided herein include: mutants comprisinga second trypsin cleavage site (i.e., NGSR (SEQ ID NO:14)) introducedinto the amino acid sequence of the fragment presented in either SEQ IDNO:6 (1218-1) or SEQ ID NO:16 (1218-1A) or the fragment presented in SEQID NO:20 (49PVD). Mutants that comprise a chymotrypsin cleavage sitecomprising either the amino acid sequence LKMS (SEQ ID NO:26) or LRMS(SEQ ID NO:48) introduced in front of (e.g., directly 5′ of) the trypsincleavage site that is naturally present in the modified polypeptidesequence; and replacement mutants in which the native trypsin site thatoccurs in the toxin domain of the modified polypeptide is destroyed anda chymotrypsin site (e.g., LKMS or LRMS) is introduced in its place.

[0095] The 1218-1 series of mutants disclosed herein are referred to asNGSR.N1218-1, LKMS.N1218-1, LKMS.R1218-1, LRMS.N1218-1, andLRMS.R1218-1. The amino acid sequences of these mutant polypeptides areset forth in SEQ ID NOS: 12, 22, 24, 42, and 44 respectively. Theinvention also provides a second series of mutant polypeptides (SEQ IDNOS:30, 32, 34, 42, and 46) in which the above-described addition(trypsin or chymotrypsin cleavage sites) and replacement (a chymotrypsincleavage site instead of the trypsin site) mutations were introducedinto the truncated polypeptide (e.g., 49PVD) set forth in SEQ ID NO: 20.This series of mutants are referred to as NGSR.N49PVD, LKMS.N49PVD,LKMS.R49PVD, LRMS.N49PVD, and LRMS.R49PVD. The amino acid sequences ofeach of the 49PVD mutant polypeptides are set forth in SEQ ID NOS: 30,32, 34, 42, and 46 respectively.

[0096] The NGSR mutants disclosed herein comprise an additionaltrypsin-sensitive protease site in a region of the amino acid sequencethat encodes domain 1 of the polypeptide. For example, the NGSR.N1218-1mutant comprises an NGSR sequence introduced between amino acid residues164 and 165 of the wild-type protein. This amino acid sequence providesa second trypsin-sensitive cleavage site into the mutant endotoxinencoded by SEQ ID NO:11. More specifically, the NGSR (e.g., SEQ IDNO:14) sequence duplicates the endogenous trypsin cleavage site that ispresent at the target location, thereby introducing a secondprotease-sensitive sight into the loop region located between alphahelices 3 and 4 of domain 1. Thus, the amino acid sequence of SEQ IDNO:14, beginning at residue 160, reads NGSRNGSR. In contrast, amino acidpositions 160-164 of the wild-type protein comprise the sequence NGSR.

[0097] While not bound by theory, it is believed that the presence of asecond protease-sensitive (e.g., trypsin or chymotrypsin) sitefacilitates intramolecular proteolytic cleavage by enhancing the abilityof helices 4 and 5 to separate from the rest of the toxin. The effectsof enhancing the ability of helices 4 and 5 to separate from the rest ofthe toxin would be manifest as a more efficient pore-forming process andhence confer an increase in the insecticidal activity of the toxin.Indeed, the Cry8 mutants described herein show improved toxicity towardsseveral Coleopteran pests. The data further suggests that the presenceof the second protease-sensitive site produces a polypeptide that ismore amenable to activation by the digestive processes of susceptibleinsects.

[0098] The mutagenized Cry8-like nucleotide sequences of the inventionmay be modified so as to change about 1, 2, 3, 4, 5, 6, 8, 10, 12 ormore of the amino acids present in the primary sequence of the encodedpolypeptide. Alternatively even more changes from the native sequencemay be introduced, such that the encoded protein may have at least about1% or 2%, or alternatively about 3% or about 4%, or even about 5% ormore of the codons altered, or otherwise modified. It should beunderstood that the mutagenized Cry8-like nucleotide sequences of thepresent invention are intended to encompass biologically functional,equivalent peptides. Such sequences may arise as a consequence of codonredundancy and functional equivalency that are known to occur naturallywithin nucleic acid sequences and the proteins thus encoded.

[0099] One of skill in the art would recognize that amino acid additionsand/or substitutions are generally based on the relative similarity ofthe amino acid side-chain substituents, for example, theirhydrophobicity, charge, size, and the like. Exemplary substitutions thattake various of the foregoing characteristics into consideration arewell known to those of skill in the art and include: arginine andlysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine, and isoleucine.

[0100] Guidance as to appropriate amino acid substitutions that do notaffect biological activity of the protein of interest may be found inthe model of Dayhoff et al. (1978) Atlas of Protein Sequence andStructure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may bepreferred.

[0101] Thus, the genes and nucleotide sequences of the invention includeboth the naturally occurring sequences as well as mutant forms.Likewise, the proteins of the invention encompass both naturallyoccurring proteins as well as variations (e.g., truncated polypeptides)and modified (e.g., mutant) forms thereof. Such variants will continueto possess the desired pesticidal activity. Obviously, the mutationsthat will be made in the DNA encoding the variant must not place thesequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

[0102] The deletions, insertions, and substitutions of the proteinsequences encompassed herein are not expected to produce radical changesin the characteristics of the protein. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by routine screening assays, such asinsect-feeding assays. See, for example, Marrone et al. (1985) J. Econ.Entomol. 78:290-293 and Czapla and Lang (1990) J. Econ. Entomol.83:2480-2485, herein incorporated by reference.

[0103] Variant nucleotide sequences and proteins also encompasssequences and proteins derived from a mutagenic and recombinogenicprocedure such as DNA shuffling. With such a procedure, one or moredifferent Cry8-like coding sequences can be manipulated to create a newpesticidal protein possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, full-length codingsequences, sequence motifs encoding a domain of interest, or anyfragment of a nucleotide sequences of the invention may be shuffledbetween the Cry8-like nucleotide sequences of the invention andcorresponding portions of other known Cry nucleotide sequences to obtaina new gene coding for a protein with an improved property of interest.

[0104] Properties of interest include, but are not limited to,pesticidal activity per unit of pesticidal protein, protein stability,and toxicity to non-target species particularly humans, livestock, andplants and microbes that express the pesticidal polypeptides of theinvention. The invention is not bound by a particular shufflingstrategy, only that at least one nucleotide sequence of the invention,or part thereof, is involved in such a shuffling strategy. Shuffling mayinvolve only nucleotide sequences disclosed herein or may additionallyinvolve shuffling of any other nucleotide sequences known in the artincluding, but not limited to, GenBank Accession Nos. U04364, U04365,and U04366. Strategies for DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0105] The nucleotide sequences of the invention can also be used toisolate corresponding sequences from other organisms, particularly otherbacteria, and more particularly other Bacillus strains. In this manner,methods such as PCR, hybridization, and the like can be used to identifysuch sequences based on their sequence homology to the sequences setforth herein. Sequences isolated based on their sequence identity to theentire Cry8-like sequences set forth herein or to fragments thereof areencompassed by the present invention. Such sequences include sequencesthat are orthologs of the disclosed sequences. By “orthologs” isintended genes derived from a common ancestral gene and which are foundin different species as a result of speciation. Genes found in differentspecies are considered orthologs when their nucleotide sequences and/ortheir encoded protein sequences share substantial identity as definedelsewhere herein. Functions of orthologs are often highly conservedamong species.

[0106] In a PCR approach, oligonucleotide primers can be designed foruse in PCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

[0107] In hybridization techniques, all or part of a known nucleotidesequence is used as a probe that selectively hybridizes to othercorresponding nucleotide sequences present in a population of clonedgenomic DNA fragments or cDNA fragments (i.e., genomic or cDNAlibraries) from a chosen organism. The hybridization probes may begenomic DNA fragments, cDNA fragments, RNA fragments, or otheroligonucleotides, and may be labeled with a detectable group such as³²P, or any other detectable marker. Thus, for example, probes forhybridization can be made by labeling synthetic oligonucleotides basedon the Cry8-like sequences of the invention. Methods for preparation ofprobes for hybridization and for construction of cDNA and genomiclibraries are generally known in the art and are disclosed in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., ColdSpring Harbor Laboratory Press, Plainview, N.Y.).

[0108] For example, an entire Cry8-like sequence disclosed herein, orone or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding Cry8-like sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique amongCry8-like sequences and are preferably at least about 10 nucleotides inlength, and most preferably at least about 20 nucleotides in length.Such probes may be used to amplify corresponding Cry8-like sequencesfrom a chosen organism by PCR. This technique may be used to isolateadditional coding sequences from a desired organism or as a diagnosticassay to determine the presence of coding sequences in a an organism.Hybridization techniques include hybridization screening of plated DNAlibraries (either plaques or colonies; see, for example, Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.).

[0109] Hybridization of such sequences may be carried out understringent conditions. By “stringent conditions” or “stringenthybridization conditions” is intended conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

[0110] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. The duration ofhybridization is generally less than about 24 hours, usually about 4 toabout 12 hours.

[0111] Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, %GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.). Thus, isolated sequences that encode aCry8-like protein of the invention and hybridize under stringentconditions to the Cry8-like sequences disclosed herein, or to fragmentsthereof, are encompassed by the present invention.

[0112] The following terms are used to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity”.

[0113] (a) As used herein, “reference sequence” is a defined sequenceused as a basis for sequence comparison. A reference sequence may be asubset or the entirety of a specified sequence; for example, as asegment of a full-length cDNA or gene sequence, or the complete cDNA orgene sequence.

[0114] (b) As used herein, “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

[0115] Methods of alignment of sequences for comparison are well knownin the art. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0116] Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, word length=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seehttp://www.ncbi.hlm.nih.gov. Alignment may also be performed manually byinspection.

[0117] Unless otherwise stated, nucleotide sequence identity/similarityvalues provided herein refer to the value obtained using GAP Version 10using the following parameters: % identity using GAP Weight of 50 andLength Weight of 3; % similarity using Gap Weight of 12 and LengthWeight of 4, or any equivalent program. For amino acid sequences, aminoacid sequence identity values provided herein refer to the valueobtained using GAP Version 10 using the following parameters: % identityusing GAP Weight of 8 and Length Weight of 2, or any equivalent program.By “equivalent program” is intended any sequence comparison programthat, for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by the preferred program.

[0118] GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

[0119] GAP presents one member of the family of best alignments. Theremay be many members of this family, but no other member has a betterquality. GAP displays four figures of merit for alignments: Quality,Ratio, Identity, and Similarity. The Quality is the metric maximized inorder to align the sequences. Ratio is the quality divided by the numberof bases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

[0120] For purposes of the present invention, comparison of nucleotideor protein sequences for determination of percent sequence identity tothe Cry8-like sequences disclosed herein is preferably made using theGAP program in the Wisconsin Genetics Software Package (Version 8 orlater) or any equivalent program. For GAP analyses of nucleotidesequences, a GAP Weight of 50 and a Length of 3 was used.

[0121] (c) As used herein, “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences makes reference tothe residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

[0122] (d) As used herein, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

[0123] (e)(i) The term “substantial identity” of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 70% sequence identity, preferably at least 80%, more preferably atleast 90%, and most preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 60%, morepreferably at least 70%, 80%, 90%, and most preferably at least 95%.

[0124] Another indication that nucleotide sequences are substantiallyidentical is if two molecules hybridize to each other under stringentconditions. Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

[0125] (e)(ii) The term “substantial identity” in the context of apeptide indicates that a peptide comprises a sequence with at least 70%sequence identity to a reference sequence, preferably 80%, morepreferably 85%, most preferably at least 90% or 95% sequence identity tothe reference sequence over a specified comparison window. Preferably,optimal alignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453. An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

[0126] The use of the term “nucleotide constructs” herein is notintended to limit the present invention to nucleotide constructscomprising DNA. Those of ordinary skill in the art will recognize thatnucleotide constructs, particularly polynucleotides andoligonucleotides, comprised of ribonucleotides and combinations ofribonucleotides and deoxyribonucleotides may also be employed in themethods disclosed herein. The nucleotide constructs, nucleic acid, andnucleotide sequences of the invention additionally encompass allcomplementary forms of such constructs, molecules, and sequences.Further, the nucleotide constructs, nucleotide molecules, and nucleotidesequences of the present invention encompass all nucleotide constructs,molecules, and sequences which can be employed in the methods of thepresent invention for transforming plants including, but not limited to,those comprised of deoxyribonucleotides, ribonucleotides, andcombinations thereof. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thenucleotide constructs, nucleic acids, and nucleotide sequences of theinvention also encompass all forms of nucleotide constructs including,but not limited to, single-stranded forms, double-stranded forms,hairpins, stem-and-loop structures, and the like.

[0127] A further embodiment of the invention relates to a transformedorganism, preferably a transformed organism selected from the groupconsisting of plant and insect cells, bacteria, yeast, baculoviruses,protozoa, nematodes, and algae, comprising a DNA molecule of theinvention, an expression cassette comprising the said DNA molecule, or avector comprising the said expression cassette, preferably stablyincorporated into the genome of the transformed organism.

[0128] The Cry8-like sequences of the invention are provided inexpression cassettes for expression in the organism of interest. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa Cry8-like sequence of the invention. By “operably linked” is intendeda functional linkage between a promoter and a second sequence, whereinthe promoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

[0129] Such an expression cassette is provided with a plurality ofrestriction sites for insertion of the Cry8-like sequence to be underthe transcriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

[0130] The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aCry8-like DNA sequence of the invention, and a transcriptional andtranslational termination region functional in the organism serving as ahost. The transcriptional initiation region, the promoter, may be nativeor analogous or foreign or heterologous to the host organism.Additionally, the promoter may be the natural sequence or alternativelya synthetic sequence. By “foreign” is intended that the transcriptionalinitiation region is not found in the native organism into which thetranscriptional initiation region is introduced. As used herein, achimeric gene comprises a coding sequence operably linked to atranscription initiation region that is heterologous to the codingsequence.

[0131] The termination region may be native with the transcriptionalinitiation region, may be native with the operably linked DNA sequenceof interest, or may be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0132] Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. For example, although nucleic acid sequencesof the present invention may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific codon preferences and GC content preferences ofmonocotyledons or dicotyledons as these preferences have been shown todiffer (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, themaize-preferred codon for a particular amino acid may be derived fromknown gene sequences from maize. Maize codon usage for 28 genes frommaize plants are listed in Table 4 of Murray et al., supra. Methods areavailable in the art for synthesizing plant-preferred genes. See, forexample, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al.(1989) Nucleic Acids Res. 17:477-498; herein incorporated by reference.

[0133] Additional sequence modifications are known to enhance geneexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon-intron splice sitesignals, transposon-like repeats, and other such well-characterizedsequences that may be deleterious to gene expression. The G-C content ofthe sequence may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. By “host cell” is meant a cell which contains a vector andsupports the replication and/or expression of the expression vector.Host cells may be prokaryotic cells such as E. coli, or eukaryotic cellssuch as yeast, insect, amphibian, or mammalian cells. Preferably, hostcells are monocotyledonous or dicotyledenous plant cells. A particularlypreferred monocotolydenous host cell is a maize host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

[0134] The expression cassettes may additionally contain 5′ leadersequences in the expression cassette construct. Such leader sequencescan act to enhance translation. Translation leaders are known in the artand include: picomavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology154:9-20), and human immunoglobulin heavy-chain binding protein (BiP)(Macejak et al. (1991) Nature 353:90-94); untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie etal. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp.237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al.(1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

[0135] In preparing the expression cassette, the various DNA fragmentsmay be manipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

[0136] A number of promoters can be used in the practice of theinvention. The promoters can be selected based on the desired outcome.The nucleic acids can be combined with constitutive, tissue-preferred,inducible, or other promoters for expression in the host organism.Suitable constitutive promoters for use in a plant host cell include,for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171);ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 andChristensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last etal. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0137] Depending on the desired outcome, it may be beneficial to expressthe gene from an inducible promoter. Of particular interest forregulating the expression of the nucleotide sequences of the presentinvention in plants are wound-inducible promoters. Such wound-induciblepromoters, may respond to damage caused by insect feeding, and includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

[0138] Additionally, pathogen-inducible promoters may be employed in themethods and nucleotide constructs of the present invention. Suchpathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. PlantPathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and VanLoon (1985) Plant Mol. Virol. 4:111-116. See also WO 99/43819, hereinincorporated by reference.

[0139] Of interest are promoters that are expressed locally at or nearthe site of pathogen infection. See, for example, Marineau et al. (1987)Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

[0140] Chemical-regulated promoters can be used to modulate theexpression of a gene in a plant through the application of an exogenouschemical regulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

[0141] Tissue-preferred promoters can be utilized to target enhancedpesticidal protein expression within a particular plant tissue.Tissue-preferred promoters include those discussed in Yamamoto et al.(1997) Plant J. 12(2)255-265; Kawamata et al. (1997) Plant Cell Physiol.38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343;Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al.(1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) PlantPhysiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol.112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

[0142] Leaf-specific promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0143] Root-specific promoters are known and can be selected from themany available from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume andersonii and the relatednon-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179.

[0144] “Seed-preferred” promoters include both “seed-specific” promoters(those promoters active during seed development such as promoters ofseed storage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase); and celA (cellulose synthase) (see WO 00/11177, hereinincorporated by reference). Gama-zein is a preferred endosperm-specificpromoter. Glob-1 is a preferred embryo-specific promoter. For dicots,seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference.

[0145] Where low level expression is desired, weak promoters will beused. Generally, by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By low level is intendedat levels of about 1/1000 transcripts to about 1/100,000 transcripts toabout 1/500,000 transcripts. Alternatively, it is recognized that weakpromoters also encompasses promoters that are expressed in only a fewcells and not in others to give a total low level of expression. Where apromoter is expressed at unacceptably high levels, portions of thepromoter sequence can be deleted or modified to decrease expressionlevels.

[0146] Such weak constitutive promoters include, for example the corepromoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No.6,072,050), the core 35S CaMV promoter, and the like. Other constitutivepromoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and6,177,611; herein incorporated by reference.

[0147] Generally, the expression cassette will comprise a selectablemarker gene for the selection of transformed cells. Selectable markergenes are utilized for the selection of transformed cells or tissues.Marker genes include genes encoding antibiotic resistance, such as thoseencoding neomycin phosphotransferase II (NEO) and hygromycinphosphotransferase (HPT), as well as genes conferring resistance toherbicidal compounds, such as glufosinate ammonium, bromoxynil,imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally,Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al.(1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al.(1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566;Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Aci. USA86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference.

[0148] The above list of selectable marker genes is not meant to belimiting. Any selectable marker gene can be used in the presentinvention.

[0149] Transformation protocols as well as protocols for introducingnucleotide sequences into plants may vary depending on the type of plantor plant cell, i.e., monocot or dicot, targeted for transformation.Suitable methods of introducing nucleotide sequences into plant cellsand subsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particleacceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050;Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No.5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment, ” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabeet al. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO00/28058). For potato transformation see Tu et al. (1998) PlantMolecular Biology 37:829-838 and Chong et al. (2000) Transgenic Research9:71-78. Additional transformation procedures can be found in Weissingeret al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York),pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

[0150] The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure expression of the desired phenotypiccharacteristic has been achieved.

[0151] The nucleotide sequences of the invention may be provided to theplant by contacting the plant with a virus or viral nucleic acids.Generally, such methods involve incorporating the nucleotide constructof interest within a viral DNA or RNA molecule. It is recognized thatthe recombinant proteins of the invention may be initially synthesizedas part of a viral polyprotein, which later may be processed byproteolysis in vivo or in vitro to produce the desired pesticidalprotein. It is also recognized that such a viral polyprotein, comprisingat least a portion of the amino acid sequence of a pesticidal protein ofthe invention, may have the desired pesticidal activity. Such viralpolyproteins and the nucleotide sequences that encode for them areencompassed by the present invention. Methods for providing plants withnucleotide constructs and producing the encoded proteins in the plants,which involve viral DNA or RNA molecules are known in the art. See, forexample, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and5,316,931; herein incorporated by reference.

[0152] The invention further relates to plant propagating material of atransformed plant of the invention including, but not limited to, seeds,tubers, corms, bulbs, leaves, and cuttings of roots and shoots.

[0153] The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosaliva), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

[0154] Vegetables include tomatoes (Lycopersicon esculentum), lettuce(e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans(Phaseolus limensis), peas (Lathyrus spp.), and members of the genusCucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis),and musk melon (C. melo). Ornamentals include azalea (Rhododendronspp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscusrosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthuscaryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Preferably, plants of the presentinvention are crop plants (for example, corn, alfalfa, sunflower,Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet,tobacco, etc.), more preferably corn and soybean plants, yet morepreferably corn plants.

[0155] Plants of particular interest include grain plants that provideseeds of interest, oil-seed plants, and leguminous plants. Seeds ofinterest include grain seeds, such as corn, wheat, barley, rice,sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean,safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax,castor, olive etc. Leguminous plants include beans and peas. Beansinclude guar, locust bean, fenugreek, soybean, garden beans, cowpea,mungbean, lima bean, fava bean, lentils, chickpea, etc.

[0156] Before plant propagation material (fruit, tuber, bulb, corm,grains, seed), but especially seed, is sold as a commercial product, itis customarily treated with a protectant coating comprising herbicides,insecticides, fungicides, bactericides, nematicides, molluscicides, ormixtures of several of these preparations, if desired together withfurther carriers, surfactants, or application-promoting adjuvantscustomarily employed in the art of formulation to provide protectionagainst damage caused by bacterial, fungal, or animal pests. In order totreat the seed, the protectant coating may be applied to the seedseither by impregnating the tubers or grains with a liquid formulation orby coating them with a combined wet or dry formulation. In addition, inspecial cases, other methods of application to plants are possible,e.g., treatment directed at the buds or the fruit.

[0157] The plant seed of the invention comprising a DNA moleculecomprising a nucleotide sequence encoding a pesticidal protein of theinvention may be treated with a seed protectant coating comprising aseed treatment compound, such as, for example, captan, carboxin, thiram,methalaxyl, pirimiphos-methyl, and others that are commonly used in seedtreatment. In one embodiment within the scope of the invention, a seedprotectant coating comprising a pesticidal composition of the inventionis used alone or in combination with one of the seed protectant coatingscustomarily used in seed treatment.

[0158] It is recognized that the genes encoding the pesticidal proteinscan be used to transform insect pathogenic organisms. Such organismsinclude Baculoviruses, fungi, protozoa, bacteria, and nematodes.

[0159] A gene encoding a pesticidal protein of the invention may beintroduced via a suitable vector into a microbial host, and said hostapplied to the environment, or to plants or animals. The term“introduced” in the context of inserting a nucleic acid into a cell,means “transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid into a eukaryotic orprokaryotic cell where the nucleic acid may be incorporated into thegenome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrialDNA), converted into an autonomous replicon, or transiently expressed(e.g., transfected mRNA).

[0160] Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest may be selected. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the pesticidalprotein, and desirably, provide for improved protection of the pesticidefrom environmental degradation and inactivation.

[0161] Such microorganisms include bacteria, algae, and fungi. Ofparticular interest are microorganisms such as bacteria, e.g.,Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces,Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes,fungi, particularly yeast, e.g., Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,Clavibacter xyli and Azotobacter vinlandir and phytosphere yeast speciessuch as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans. Of particularinterest are the pigmented microorganisms.

[0162] A number of ways are available for introducing a gene expressingthe pesticidal protein into the microorganism host under conditions thatallow for stable maintenance and expression of the gene. For example,expression cassettes can be constructed which include the nucleotideconstructs of interest operably linked with the transcriptional andtranslational regulatory signals for expression of the nucleotideconstructs, and a nucleotide sequence homologous with a sequence in thehost organism, whereby integration will occur, and/or a replicationsystem that is functional in the host, whereby integration or stablemaintenance will occur.

[0163] Transcriptional and translational regulatory signals include, butare not limited to, promoters, transcriptional initiation start sites,operators, activators, enhancers, other regulatory elements, ribosomalbinding sites, an initiation codon, termination signals, and the like.See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2;Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed.Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.); Davis et al., eds. (1980) Advanced Bacterial Genetics(Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y.; and thereferences cited therein.

[0164] Suitable host cells, where the pesticidal protein-containingcells will be treated to prolong the activity of the pesticidal proteinsin the cell when the treated cell is applied to the environment of thetarget pest(s), may include either prokaryotes or eukaryotes, normallybeing limited to those cells that do not produce substances toxic tohigher organisms, such as mammals. However, organisms that producesubstances toxic to higher organisms could be used, where the toxin isunstable or the level of application sufficiently low as to avoid anypossibility of toxicity to a mammalian host. As hosts, of particularinterest will be the prokaryotes and the lower eukaryotes, such asfungi. Illustrative prokaryotes, both Gram-negative and gram-positive,include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella,Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium;Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas,Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae,such as Pseudomonas and Acetobacter; Azotobacteraceae andNitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes andAscomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

[0165] Characteristics of particular interest in selecting a host cellfor purposes of pesticidal protein production include ease ofintroducing the pesticidal protein gene into the host, availability ofexpression systems, efficiency of expression, stability of the proteinin the host, and the presence of auxiliary genetic capabilities.Characteristics of interest for use as a pesticide microcapsule includeprotective qualities for the pesticide, such as thick cell walls,pigmentation, and intracellular packaging or formation of inclusionbodies; leaf affinity; lack of mammalian toxicity; attractiveness topests for ingestion; ease of killing and fixing without damage to thetoxin; and the like. Other considerations include ease of formulationand handling, economics, storage stability, and the like.

[0166] Host organisms of particular interest include yeast, such asRhodotorula sp., Aureobasidium sp., Saccharomyces sp., andSporobolomyces sp., phylloplane organisms such as Pseudomonas sp.,Erwinia Sp., and Flavobacterium sp., and other such organisms, includingPseudomonas aeurginosa, Pseudomonas fluorescens, Saccharomycescerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis,and the like.

[0167] Genes encoding the pesticidal proteins of the invention can beintroduced into microorganisms that multiply on plants (epiphytes) todeliver pesticidal proteins to potential target pests. Epiphytes, forexample, can be gram-positive or gram-negative bacteria.

[0168] Root-colonizing bacteria, for example, can be isolated from theplant of interest by methods known in the art. Specifically, a Bacilluscereus strain that colonizes roots can be isolated from roots of a plant(see, for example, Handelsman et al. (1991) Appl. Environ. Microbiol.56:713-718). Genes encoding the pesticidal proteins of the invention canbe introduced into a root-colonizing Bacillus cereus by standard methodsknown in the art.

[0169] Genes encoding pesticidal proteins can be introduced, forexample, into the root-colonizing Bacillus by means ofelectrotransformation. Specifically, genes encoding the pesticidalproteins can be cloned into a shuttle vector, for example, pHT3101(Lerecius et al. (1989) FEMS Microbiol. Letts. 60:211-218. The shuttlevector pHT3101 containing the coding sequence for the particularpesticidal protein gene can, for example, be transformed into theroot-colonizing Bacillus by means of electroporation (Lerecius et al.(1989) FEMS Microbiol. Letts. 60:211-218).

[0170] Expression systems can be designed so that pesticidal proteinsare secreted outside the cytoplasm of gram-negative bacteria, E. coli,for example. Advantages of having pesticidal proteins secreted are: (1)avoidance of potential cytotoxic effects of the pesticidal proteinexpressed, and (2) improvement in the efficiency of purification of thepesticidal protein, including, but not limited to, increased efficiencyin the recovery and purification of the protein per volume cell brothand decreased time and/or costs of recovery and purification per unitprotein.

[0171] Pesticidal proteins can be made to be secreted in E. coli, forexample, by fusing an appropriate E. coli signal peptide to theamino-terminal end of the pesticidal protein. Signal peptides recognizedby E. coli can be found in proteins already known to be secreted in E.coli, for example the OmpA protein (Ghrayeb et al (1984) EMBO J,3:2437-2442). OmpA is a major protein of the E. coli outer membrane, andthus its signal peptide is thought to be efficient in the translocationprocess. Also, the OmpA signal peptide does not need to be modifiedbefore processing as may be the case for other signal peptides, forexample lipoprotein signal peptide (Duffaud et al. (1987) Meth. Enzymol.153:492).

[0172] Pesticidal proteins of the invention can be fermented in abacterial host and the resulting bacteria processed and used as amicrobial spray in the same manner that Bacillus thuringiensis strainshave been used as insecticidal sprays. In the case of a pesticidalprotein(s) that is secreted from Bacillus, the secretion signal isremoved or mutated using procedures known in the art. Such mutationsand/or deletions prevent secretion of the pesticidal protein(s) into thegrowth medium during the fermentation process. The pesticidal proteinsare retained within the cell, and the cells are then processed to yieldthe encapsulated pesticidal proteins. Any suitable microorganism can beused for this purpose. Pseudomonas has been used to express Bacillusthuringiensis endotoxins as encapsulated proteins and the resultingcells processed and sprayed as an insecticide (Gaertner et al. (1993),in: Advanced Engineered Pesticides, ed. Kim).

[0173] Alternatively, the pesticidal proteins are produced byintroducing a heterologous gene into a cellular host. Expression of theheterologous gene results, directly or indirectly, in the intracellularproduction and maintenance of the pesticide. These cells are thentreated under conditions that prolong the activity of the toxin producedin the cell when the cell is applied to the environment of targetpest(s). The resulting product retains the toxicity of the toxin. Thesenaturally encapsulated pesticidal proteins may then be formulated inaccordance with conventional techniques for application to theenvironment hosting a target pest, e.g., soil, water, and foliage ofplants. See, for example EPA 0192319, and the references cited therein.

[0174] In the present invention, a transformed microorganism, whichincludes whole organisms, cells, spore(s), pesticidal protein(s),pesticidal component(s), pest-impacting component(s), mutant(s);preferably living or dead cells and cell components, including mixturesof living and dead cells and cell components, and including broken cellsand cell components, or an isolated pesticidal protein, can beformulated with an acceptable carrier into a pesticidal composition(s)that is, for example, a suspension, a solution, an emulsion, a dustingpowder, a dispersible granule, a wettable powder, and an emulsifiableconcentrate, an aerosol, an impregnated granule, an adjuvant, a coatablepaste, and also encapsulations in, for example, polymer substances.

[0175] Such compositions disclosed above may be obtained by the additionof a surface-active agent, an inert carrier, a preservative, ahumectant, a feeding stimulant, an attractant, an encapsulating agent, abinder, an emulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematocides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular target pests.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders, or fertilizers. The activeingredients of the present invention are normally applied in the form ofcompositions and can be applied to the crop area or plant to be treated,simultaneously or in succession, with other compounds. Methods ofapplying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the pesticidal proteins produced by the bacterial strains of thepresent invention include, but are not limited to, foliar application,seed coating, and soil application. The number of applications and therate of application depend on the intensity of infestation by thecorresponding pest.

[0176] Suitable surface-active agents include, but are not limited to,anionic compounds such as a carboxylate of, for example, a metal;carboxylate of a long chain fatty acid; an N-acylsarcosinate; mono ordi-esters of phosphoric acid with fatty alcohol ethoxylates or salts ofsuch esters; fatty alcohol sulfates such as sodium dodecyl sulfate,sodium octadecyl sulfate or sodium cetyl sulfate; ethoxylated fattyalcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates;petroleum sulfonates; alkyl aryl sulfonates such as alkyl-benzenesulfonates or lower alkylnaphtalene sulfonates, e.g., butyl-naphthalenesulfonate; salts of sulfonated naphthalene-formaldehyde condensates;salts of sulfonated phenol-formaldehyde condensates; more complexsulfonates such as the amide sulfonates, e.g., the sulfonatedcondensation product of oleic acid and N-methyl taurine; or the dialkylsulfosuccinates, e.g., the sodium sulfonate or dioctyl succinate.Non-ionic agents include condensation products of fatty acid esters,fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substitutedphenols with ethylene oxide, fatty esters of polyhydric alcohol ethers,e.g., sorbitan fatty acid esters, condensation products of such esterswith ethylene oxide, e.g., polyoxyethylene sorbitar fatty acid esters,block copolymers of ethylene oxide and propylene oxide, acetylenicglycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylatedacetylenic glycols. Examples of a cationic surface-active agent include,for instance, an aliphatic mono-, di, or polyamine such as an acetate,naphthenate or oleate; or oxygen-containing amine such as an amine oxideof polyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

[0177] Examples of inert materials include but are not limited toinorganic minerals such as kaolin, phyllosilicates, carbonates,sulfates, phosphates, or botanical materials such as cork, powderedcorncobs, peanut hulls, rice hulls, and walnut shells.

[0178] The compositions of the present invention can be in a suitableform for direct application or as a concentrate of primary compositionthat requires dilution with a suitable quantity of water or otherdiluant before application. The pesticidal concentration will varydepending upon the nature of the particular formulation, specifically,whether it is a concentrate or to be used directly. The compositioncontains 1 to 98% of a solid or liquid inert carrier, and 0 to 50%,preferably 0.1 to 50% of a surfactant. These compositions will beadministered at the labeled rate for the commercial product, preferablyabout 0.01 lb-5.0 lb. per acre when in dry form and at about 0.01pts.-10 pts. per acre when in liquid form.

[0179] In a further embodiment, the compositions, as well as thetransformed microorganisms and pesticidal proteins, of the invention canbe treated prior to formulation to prolong the pesticidal activity whenapplied to the environment of a target pest as long as the pretreatmentis not deleterious to the activity. Such treatment can be by chemicaland/or physical means as long as the treatment does not deleteriouslyaffect the properties of the composition(s). Examples of chemicalreagents include but are not limited to halogenating agents; aldehydessuch a formaldehyde and glutaraldehyde; anti-infectives, such aszephiran chloride; alcohols, such as isopropanol and ethanol; andhistological fixatives, such as Bouin's fixative and Helly's fixative(see, for example, Humason, (1967) Animal Tissue Techniques (W. H.Freeman and Co.).

[0180] In other embodiments of the invention, it may be advantageous totreat the Cry8-like polypeptides with a protease, for example trypsin,to activate the protein prior to application of a pesticidal proteincomposition of the invention to the environment of the target pest.Methods for the activation of protoxin by a serine protease are wellknown in the art. See, for example, Cooksey (1968) Biochem. J. 6:445-454and Carroll and Ellar (1989) Biochem. J. 261:99-105, the teachings ofwhich are herein incorporated by reference. For example, a suitableactivation protocol includes, but is not limited to, combining apolypeptide to be activated, for example a purified 1218-1 polypeptide,and trypsin at a 1/100 weight ratio 1218-1 protein/trypsin in 20 nMNaHCO3, pH 8 and digesting the sample at 36° C. for 3 hours.

[0181] The compositions, as well as the transformed microorganisms andpesticidal proteins, of the invention can be applied to the environmentof an insect pest by, for example, spraying, atomizing, dusting,scattering, coating or pouring, introducing into or on the soil,introducing into irrigation water, by seed treatment or generalapplication or dusting at the time when the pest has begun to appear orbefore the appearance of pests as a protective measure. It is generallyimportant to obtain good control of pests in the early stages of plantgrowth, as this is the time when the plant can be most severely damaged.The compositions of the invention can conveniently contain anotherinsecticide if this is thought necessary. In an embodiment of theinvention, the composition is applied directly to the soil, at a time ofplanting, in granular form of a composition of a carrier and dead cellsof a Bacillus strain or transformed microorganism of the invention.Another embodiment is a granular form of a composition comprising anagrochemical such as, for example, a herbicide, an insecticide, afertilizer, an inert carrier, and dead cells of a Bacillus strain ortransformed microorganism of the invention.

[0182] The embodiments of the present invention may be effective againsta variety of pests. For purposes of the present invention, pestsinclude, but are not limited to, insects, fungi, bacteria, nematodes,acarids, protozoan pathogens, animal-parasitic liver flukes, and thelike. Preferred pests are insect pests, particularly insect pests thatcause significant damage, most particularly insect pests that causesignificant damage to agricultural plants. By “insect pests” is intendedinsects and other similar pests such as, for example, those of the orderAcari including, but not limited to, mites and ticks. Insect pests ofthe present invention include, but are not limited to, insects of theorder Lepidoptera, e.g. Achoroia grisella, Acleris gloverana, Aclerisvariana, Adoxophyes orana, Agrotis ipsilon, Alabama argillacea,Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsialineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis,Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrixthurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Coliaseurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella,Datana integerrima, Dendrolimus sibericus, Desmia feneralis, Diaphaniahyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraeasaccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia elutella,Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoelliaambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoamessoria, Galleria mellonella, Grapholita molesta, Harrisina americana,Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileucaoliviae, Homoeosoma electellum, Hyphantia cunea, Keiferialycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellarialugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis,Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestrabrassicae, Mamestra configurata, Manduca quinquemaculata, Manduca sexta,Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp.,Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes,Pectinophora gossypiella, Phryganidia californica, Phyllonorycterblancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynotaflouendana, Platynota stultana, Platyptilia carduidactyla, Plodiainterpunctella, Plutella xylostella, Pontia protodice, Pseudaletiaunipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizuraconcinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp.,Thaurnstopoea pityocampa, Tinsola bisselliella, Trichoplusia hi, Udearubigalis, Xylomyges curiails, and Yponomeuta padella.

[0183] Also, the embodiments of the present invention may be effectiveagainst insect pests including insects selected from the orders Diptera,Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera,Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera,etc., particularly Coleoptera, especially Diabrotica virgifera andLepidoptera. Insect pests of the invention for the major crops include:Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, blackcutworm; Helicoverpa zea, corn earworm; Spodoptera frugiperda, fallarmyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpuslignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcaneborer; Diabrotica virgifera, western corn rootworm; Diabroticalongicornis barberi, northern corn rootworm; Diabrotica undecimpunctatahowardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn bloth leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, two spotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, lesercornstalk borer; Feltia subterranea, granulate cutworm; Phyllophagacrinita, white grub; Eleodes, Conoderus, and Aeolus spp., wireworms;Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn fleabeetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; cornleaf aphid; Sipha flava, yellow sugarcane aphid; Blissus leucopterusleucopterus, chinch bug; Contarinia sorghicola, sorghum midge;Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae,two-spotted spider mite; Wheat: Pseudaletia unipunctata, army worm;Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lessercornstalk borer; Agrotis orthogonia, pale western cutworm; Elasmopalpuslignosellus, lesser cornstalk borer; Oulema melanopus, cereal leafbeetle; Hypera punctata, clover leaf weevil; Diabrotica undecimpunctatahowardi, southern corn rootworm; Russian wheat aphid; Schizaphisgraminum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus differentialis,differential grasshopper; Melanoplus sanguinipes, migratory grasshopper;Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat midge;Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulbfly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stemsawfly; Aceria tulipae, wheat curl mite; Sunflower: Cylindrocupturusadspersus, sunflower stem weevil; Smicronyx fulus, red sunflower seedweevil; Smicronyx sordidus, gray sunflower seed weevil; Suleimahelianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth;Zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrotbeetle; Neolasioptera murtfeldtiana, sunflower seed midge; Cotton:Heliothis virescens, tobacco budworm; Helicoverpa zea, cotton bollworm;Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pinkbollworm; Anthonomus grandis grandis, boll weevil; Aphis gossypii,cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodesabutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plantbug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Thrips tabaci, onion thrips;Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, carminespider mite; Tetranychus urticae, two-spotted spider mite; Rice:Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grapecolaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilusoryzae, rice weevil; Nephotettix nigropictus, rice leafhoper; Blissusleucopterus leucopterus, chinch bug; Acrosternum hilare, green stinkbug; Soybean: Pseudoplusia includens, soybean looper; Anticarsiagemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm;Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm;Spodoptera exigua, beet armyworm; Heliothis virescens, tobacco budworm;Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican beanbeetle; Myzus persicae, green peach aphid; Empoasca fabae, potatoleafhopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum,redlegged grasshopper; Melanoplus differentialis, differentialgrasshopper; Hylemya platura, seedcorn maggot; Sericothrips variabilis,soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani,strawberry spider mite; Tetranychus urticae, two-spotted spider mite;Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, blackcutworm; Schizaphis graminum, greenbug; Blissus leucopterus leucopterus,chinch bug; Acrosternum hilare, green stink bug; Euschistus servus,brown stink bug; Jylemya platura, seedcorn maggot; Mayetiola destructor,Hessian fly; Petrobia latens, brown wheat mite; Oil Seed Rape:Vrevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, cruciferflea beetle; Potato: Leptinotarsa decemlineata, Colorado potato beetle.

[0184] Furthermore, embodiments of the present invention may beeffective against Hemiptera such as Lygus hesperus, Lygus lineolaris,Lygus pratensis, Lygus rugulipennis Popp, Lygus pabulinus, Calocorisnorvegicus, Orthops compestris, Plesiocoris rugicollis, Cyrtopeltismodestus, Cyrtopeltis notatus, Spanagonicus albofasciatus, Diaphnocorischlorinonis, Labopidicola allii, Pseudatomoscelis seriatus, Adelphocorisrapidus, Poecilocapsus lineatus, Blissus leucopterus, Nysius ericae,Nysiusraphanus, Euschistus servus, Nezara viridula, Eurygaster,Coreidae, Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, andCimicidae.

[0185] Nematodes include plant-parasitic nematodes such as root-knot,cyst, and lesion nematodes, including Heterodera and Globodera spp;particularly Globodera rostochiensis and Globodera pailida (potato cystnematodes); Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); and Heterodera avenae (cereal cystnematode).

[0186] The preferred developmental stage for testing for pesticidalactivity is larvae or immature forms of these above mentioned insectpests. The insects may be reared in total darkness at from about 20° C.to about 30° C. and from about 30% to about 70% relative humidity.Bioassays may be performed as described in Czapla and Lang (1990) J.Econ. Entomol. 83(6): 2480-2485. Methods of rearing insect larvae andperforming bioassays are well known to one of ordinary skill in the art.

[0187] A wide variety of bioassay techniques is known to one skilled inthe art. General procedures include addition of the experimentalcompound or organism to the diet source in an enclosed container.Pesticidal activity can be measured by, but is not limited to,mortality, weight loss, attraction, repellency and other behavioral andphysical changes after feeding and exposure for an appropriate length oftime. Bioassays described herein can be used with any feeding insectpest in the larval or adult stage.

[0188] The following examples are presented by way of illustration, notby way of limitation.

EXPERIMENTAL Example 1 Bioassay for Testing the Pesticidal Activity ofB. thuringiensis Strains against Western Corn Rootworm and Southern CornRootworm

[0189] Insect diets for Colorado potato beetle (CPB), southern cornrootworm (SCRW), and western corn rootworm (WCRW) larvae are known inthe art. See, for example, Rose and McCabe (1973) J. Econ. Entomology66:393, herein incorporated by reference. The insect diet is preparedand poured onto a Pittman tray. Generally 1.5 mL of diet is dispensedinto each cell with an additional 150 μL of sample preparation appliedto the diet surface.

[0190] Bacterial colonies from an original plate of transformantsexpressing the pesticidal proteins of interest are spotted on replicaplates and inoculated in 5 mL 2×YT broth with 500 μL/1000 mL kanamycinantibiotic. The tubes are grown overnight. If no growth is present, thetubes are incubated for an additional 24 hours. Following incubation,the tubes are centrifuged at 3500 rpms for 5-8 minutes. The supernatantis discarded and the pellet resuspended in 1000 μL PBS. The sample isthen transferred to 1.5 mL eppendorf tubes and incubated on ice untilthe temperature is 3 to 4° C., followed by sonication for 12-15 seconds.

[0191] Microbial culture broths (150 μL) or other samples (150 μL) areoverlayed onto 1.5 mL artificial diets with a 2.54 cm² surface area. Forthe screening of pesticidal activity against rootworms, 25 μL of a 0.8%egg agar solution is applied to lids of the trays. The trays and lidsare allowed to dry under a hood. After drying, the lids are placed ontrays and incubated for 4-7 days at a temperature of 26° C. Thebioassays are then scored by counting “live” versus “dead” larvae.Mortality is calculated as percentage of dead larvae out of the totallarvae tested.

Example 2 Pesticidal Activity of B. thuringiensis Strain 1218 Lysates

[0192] Samples prepared from cultures of B. thuringiensis strains 1218were tested for the presence of pesticidal activity against CPB, WCRW,and SCRW as described in Example 1. As a control, the diet was treatedwith phosphate-buffered saline (PBS).

[0193] To prepare each sample, an individual colony of a strain growingon an LB plate was selected and used to inoculate a tube containing 50mL of TB medium. The tube was incubated overnight at 28° C. and 250 rpm.Following the incubation, the tube was centrifuged at 4300×g for 15minutes. The supernatant was discarded and the pellet resuspended in 50mL of sporulation medium. The tube was centrifuged again at 4300×g for15 minutes. The second supernatant was discarded, and the second pelletresuspended in 50 mL of sporulation medium. The tube was then incubatedfor 48 hours at 28° C. and 250 rpm. Following this incubation, the tubewas centrifuged at 4300×g for 15 minutes. The supernatant was discarded,and the pellet was resuspended in 10 mL of 1×M9 medium. The sample wasthen transferred to a 1.5 mL microfuge tube, incubated on ice until thetemperature was about 3 to 4° C., and then sonicated for 12-15 seconds.For bioassays, 150 μL of a sonicated sample was used.

[0194] Sporulation medium comprises 200 mL of 5×M9 salts solution, 5 mLof salts solution, 5 mL of CaCl₂ solution, and dH₂0 to a final volume of1 L. The solution of 5×M9 salts comprises: 64 g, Na₂HPO₄.7H₂O; 15 g,KH₂PO₄; 2.5 g, NaCl; 5 g, NH₄Cl; and dH2O to a final volume of 1.0 L.Salts solution comprises: 2.46 g, MgSO₄.7H₂O; 0.04 g, MnSO₄.H2O; 0.28 g,ZnSO₄.7H2O; 0.40 g, FeSO₄.7H2O; and dH₂O to a final volume of 1.0 L.CaCl₂ solution comprises 3.66 g CaCl₂.2H₂O and dH2O to a final volume of100 ml.

[0195] Samples were tested with and without heating to determine whetherthe component(s) responsible for the pesticidal activity is heat stable.For the heat treatment, the samples were boiled for 15 minutes prior touse in the bioassay. Unheated samples prepared from strain 1218exhibited pesticidal activity against western corn rootworm, with lesserpesticidal activity against southern corn rootworm. The samples preparedfrom strain 1218 lysates caused moderate stunting in the southern cornrootworm larvae. Following heating, the samples had greatly reducedpesticidal activity against both species of rootworms.

[0196] The reduction in pesticidal activity following heating indicatedthat the one or more components of the sample from strain 1218 that isresponsible for the pesticidal activity is heat labile. Such a reductionis consistent with one or more of the components being a protein.

Example 3 Pesticidal Activity of Crystal Proteins Isolated from B.thuringiensis Strain 1218

[0197] Using samples of sporulated cultures of B. thuringiensis strain1218 prepared as described in Example 2, crystal proteins were isolatedand then trypsin-treated using methods known in the art. Briefly, afterpurification (zonal gradient centrifugation, Renografin-76), thepurified crystals were dissolved in alkaline buffer (50 mM Na₂CO₃, 10 mMdithiothreitol, pH 10). Prior to use in the assays, the dissolvedcrystal proteins were concentrated by filtration with Centriprep®(Millipore Corp.) centrifugal filter units with a MW cutoff of 10,000.

[0198] It is recognized that under some experimental conditions, it maybe advantageous to treat the Cry8-like polypeptides with a protease, forexample trypsin, to activate the protein prior to determining thepesticidal activity of a particular sample. Methods for the activationof protoxin by a serine protease are well known in the art. See, forexample, Cooksey (1968) Biochem J. 6:445-454 and Carroll and Ellar(1989) Biochem J. 261:99-105; herein incorporated by reference. Isolatedcrystal proteins were screened for pesticidal activity against westerncorn rootworm larvae as described in Example 1. Both a new crystalprotein preparation and a previously made preparation (“oldpreparation”) from strain 1218 possessed substantial pesticidal activityagainst western corn rootworms. Dissolved crystal proteins were storedat −80° C. for 20 days before use in the assays.

[0199] A skilled artisan will acknowledge that there are numerousindicators of pesticidal activity and that variables such as number ofdead insects, or average weight of treated insects can be monitored. Forexample, pesticidal activity can be conveniently expressed as %mortality, which is the percentage of dead rootworm larvae out of thetotal number of larvae.

Example 4 Nucleotide Sequences Isolated from B. thuringiensis Strain1218

[0200] An effort was undertaken to isolate the nucleotide sequences thatencode the crystal proteins from B. thuringiensis strain 1218. Twonucleotide sequences were isolated from 1218 that have nucleotidesequence and amino acid sequence homology to Cry8Bal (GenBank AccessionNo. U04365). The two Cry8-like coding sequences isolated from strain1218 have been designated Cry1218-1 (SEQ ID NO:1) and Cry1218-2 (SEQ IDNO:3). SEQ ID NO:27 and SEQ ID NO:28 provide the nucleic acid sequencesof native genomic clones of Cry1218-1 and Cry1218-2, respectively.

[0201] To determine if the proteins encoded by variant or mutantpolynucleotides of the invention encode proteins with pesticidalactivity, each of the nucleic acid sequence was expressed in Escherichiacoli. For example, to determine if the 1218-1 or 1218-2 polynucleotidesequences provided herein encode polypeptides with pesticidal activity,truncated nucleotide sequences were prepared. SEQ ID NO:15 correspondsto nucleotides 1 through 2007 of the nucleotide sequence of Cry1218-1(SEQ ID NO:1). SEQ ID NO:17 corresponds to nucleotides 1 through 2019 ofthe nucleotide sequence of Cry1218-2 (SEQ ID NO:3).

[0202] SEQ ID NOS:15 and 17 encode truncated Cry8-like polypeptideshaving the amino acid sequences set forth in SEQ ID NO:16 and 18,respectively. Each of the truncated nucleotide sequences (SEQ ID NOs:15and 17) was separately cloned into a pET28a expression vector and thenused to transform E. coli. Transformed colonies were selected and grownin liquid culture as described in Example 1. The expressed,N-terminal-His-tagged, truncated Cry8-like proteins were isolated fromE. coli lysates by affinity chromatography using a Nickel affinitycolumn. The column fractions with the protein of interest were dialyzedextensively against 10 mM Tris-HCl (pH 8.5) and then concentrated usingCentriprep® (Millipore Corp.) centrifugal filter units with a MW cutoffof 10,000 according to the manufacturer's directions. The concentratedCry8-like protein samples were tested for the presence of pesticidalactivity against western corn rootworm as described in Example 1.

[0203] Bioassays evaluating the pesticidal activity of recombinantCry8-like proteins purified from E. coli-expressed preparations wereconducted as described in Example 1 with the aqueous protein samplesoverlaid on the surface of the rootworm diet. The pesticidal activity ofwild-type (e.g., native) and mutant endotoxin were assessed againstsouthern corn rootworms. As expected, it was observed that thepesticidal activity decreased as the concentration of the truncatedCry8-like proteins applied to the diet decreased.

[0204] Pesticidal activity was also assessed by incorporating thepesticidal proteins into the rootworm diet, as opposed to the methoddescribed above, which involved incorporating a protein-containingsolution into the diet mixture. For example, sample diets comprising1000, 500, 400, 300, 200, or 100 ppm of a pesticidal polypeptideincorporated into the diet were assessed.

Example 5 Preparation of a Plant-Preferred Nucleotide Sequence Encodinga Pesticidal Protein

[0205] Because codon usage is different between plants and bacteria, theexpression in a plant of a protein encoded by nucleotide sequence ofbacterial origin can be limited due to translational inefficiency in theplant. It is known in the art that expression can be increased in aplant by altering the coding sequence of the protein to containplant-preferred codons. For optimal expression of a protein in a plant,a synthetic nucleotide sequence may be prepared using the amino acidsequence of the protein and back-translating the sequence usingplant-preferred codons.

[0206] Using such an approach, a portion of the amino acid sequence ofthe protein encoded by Cry1218-1 (SEQ ID NO:2) was back-translated usingmaize-preferred codons. The resulting plant-preferred nucleotidesequence is set forth in SEQ ID NO:9. The nucleotide sequence set forthin SEQ ID NO:9 encodes a polypeptide (SEQ ID NO:10) that comprises thefirst 669 amino acids of the amino acid sequence set forth in SEQ IDNO:2. Thus, SEQ ID NOS:10 and 16 encode polypeptides comprising the sameamino acid sequence, and SEQ ID NO:15 provides a second polynucleotidethat encodes the amino acid sequences set forth in SEQ ID NO:10.

Example 6 Bioassay for Testing the Pesticidal Activity of MutantCry8-like Polypeptides against Colorado Potato Beetle (Leptinotarsadecemlineata)

[0207] Protocol

[0208] Briefly, bioassay parameters were as follows: Bio-Serv diet(catalog number F9800B, from: BIOSERV, Entomology Division, One 8^(th)Street, Suite 1, Frenchtown, N.J. 08825) was dispensed in 128-wellPitman trays (catalog number BIO-BA-128 from CD International, Pitman,N.J. 08071) having a surface area of 2.4 cm². Cry 8-like samples(1218-1A, 49PVD, and NGSR1218-1) were applied topically to the dietsurface at a rate of 50 μl/well. Enough sample material was supplied toprovide for 4 observations/sample. After the sample dried, 2 Coloradopotato beetle neonates were added to each well. Therefore, there was atotal of 8 larvae/sample. A lid was placed on each tray (catalog numberBIO-CV-16, CD International, Pitman, N.J., 08071) and the trays wereplaced in an incubator at 25° C.

[0209] The assay trays showed no surface contamination present in thebuffer controls or the wells that contained Cry8-like samples. The testwas scored for mortality on the 4^(th) day following live infesting.TABLE 1 Pesticidal Activity of Truncated 1218-1 Polypeptides and aTrypsin Addition-Mutant against Colorado Potato Beetle Code SamplesProtein (mg/ml) Mortality A a-buffer 1/8 13% B b-1218-1A 0.05 7/8 88% Cc-1218-1A 0.025 7/8 88% D d-1218-1A 0.013 4/6 67% F f-49PVD 0.1 8/8 100%G g-49PVD 0.05 4/8 50% H h-49PVD 0.025 8/9 89% L l-NGSR1218-1 0.1 8/8100% M m-NGSR1218-1 0.05 8/8 100% N n-NGSR1218-1 0.025 8/8 100%

[0210] Results

[0211] The sample labeled “A” in Table 1 is a control sample consistingof 10 mM carbonate buffer at pH 10. All of the truncated and mutantprotein samples 1218-1A (b-d), 49PVD (f-h), and NGSR1218-1 (l-n) weresolubilized in 10 mM carbonate buffer at pH 10.

[0212] The 1218-1A samples, b-d, comprise a truncated polypeptidesequence comprising the amino acid sequence set forth in SEQ ID NO:16.More specifically, the 1218-1A samples comprise the truncated toxindomain represented by amino acid (aa) residue 1 to aa 669 (from M to E)of the amino acid sequences set forth in SEQ ID NO:2.

[0213] The 49PVD samples, f-h, comprise a mutant polypeptide sequencehaving an amino acid sequence that is set forth in SEQ ID NO:20. 49PVDwas generated by trimming sequence from both the N-terminus and theC-terminus of the sequence set forth in SEQ ID NO:16. More specifically,the N-terminus of the 49PVD mutant was trimmed by 47 residues; thus, thepolypeptide starts at aa residue 48(M) and the C-1218-1A (SEQ ID NO:16)from aa residue 48 to aa 663.

[0214] The NGSR samples, l-m, comprise a 1218-1 mutant polypeptidesequence that is set forth in SEQ ID NO: 12. NGSR1218-1 was generated bythe addition of an NGSR motif to the amino acid sequence set forth inSEQ ID NO:16 after aa 164. More specifically, the NGSR mutant provides a1218-1A mutant that includes the amino acid sequence NGSR between aa 164and aa 165 of the sequence set forth in SEQ ID NO:16. The addition of 4residues to 1218-1A generated a protein with 673 aa. Bioassays of1218-1A, 49PVD, and NGSR1218-1 indicated that all three protein samplesare efficacious against Colorado potato beetle (CPB). Mutant NGSR1218-1was found to be more potent that the parent 1218-1A and 49PVD mutant.The modified (e.g., truncated or mutant) 1218-1 polypeptides (49PVD,NGSR1218-1) were at least as active as the relevant 1218-1 or 1218-1Acontrol sample.

Example 7 Bioassay for Testing the Pesticidal Activity of MutantCry8-like Polypeptides against Southern Corn Rootworm and Western CornRootworm

[0215] Protocol

[0216] Briefly, the assay parameters described above in Example 6 weremodified to allow for the evaluation of the pesticidal activity ofadditional 1218-1,1218-1A or 49PVD mutants against western corn rootworm(WCRW) and southern corn rootworm (SCRW). Briefly, Bio-Serv diet(catalog number F9800B, from: BIOSERV, Entomology Division, One 8^(th)Street, Suite 1, Frenchtown, N.J. 08825) was dispensed in 128-wellPitman trays (catalog number BIO-BA-128 from CD International, Pitman,N.J. 08071) having a surface area of 2.4 cm².

[0217] Cry 8-like samples were applied topically to the diet surface ata volume of 50 μl/well. Enough sample material was supplied to providefor replicate observations/sample. For the screening of pesticidalactivity against rootworms, 25 μL of a 0.8% egg agar solution is appliedto lids of the trays. The trays and lids are allowed to dry under ahood. After drying, the lids are placed on trays and incubated for 4-7days at a temperature of 26° C. A lid was placed on each tray (catalognumber BIO-CV-16, CD International, Pitman, N.J., 08071), and the trayswere placed in an incubator at 25° C.

[0218] For the evaluation of pesticidal activity against SCRW, insectswere exposed to a solution comprising either buffer (50 mM carbonatebuffer (pH 10) or a 1218-1 or 1218-1A mutant polypeptide (e.g.,1218-1A), LKMS.N1218-1, LKMS.R1218-1, NGSR.N1218-1, LKMS.N49PVD,LKMS.R49PVD, or NGSR.N49PVD) at a doses of either 36 or 3.6 μg/cm².

[0219] For the evaluation of pesticidal activity against WCRW, insectswere exposed to a solution comprising either buffer (50 mM carbonatebuffer (pH 10) or to a limited number of the mutant 1218-1 polypeptides(LKMS.R1218-1, NGSR.N1218-1, LKMS.N49PVD, LKMS.R49PVD, or NGSR.N49PVD)at 88 μg/cm². The bioassays are then scored by counting “live” versus“dead” larvae. Mortality is calculated as percentage of dead larvae outof the total larvae tested. TABLE 2 Pesticidal Activity of Cry1218-1Mutant Polypeptides against Southern Corn Rootworm- Replicate 1 % MOR- %MOR- POLYPEPTIDE DOSE TALITY DOSE TALITY 1218-1A 36 μg/cm²  0 3.6 μg/cm²3 LKMS.N 1218-1 36 μg/cm²  6 3.6 μg/cm² 4 (no protein) LKMS.R 1218-1 36μg/cm² 89 3.6 μg/cm² 27 NGSR.N 1218-1 36 μg/cm² 80 3.6 μg/cm² 8 50 mMCarbonate —  0 — 0 Buffer (pH10) 49PVD 36 μg/cm²  3 3.6 μg/cm² 3LKMS.N49PVD 36 μg/cm² 69 3.6 μg/cm² 11 LKMS.R49PVD 36 μg/cm² 60 3.6μg/cm² 17 NGSR.N49PVD 36 μg/cm² 93 3.6 μg/cm² 22

[0220] TABLE 3 Pesticidal Activity of Cry1218-1 Mutant Polypeptidesagainst Southern Corn Rootworm- Replicate 2 % MOR- % MOR- POLYPEPTIDEDOSE TALITY DOSE TALITY 1218-1A 36 μg/cm² 3 3.6 μg/cm² 0 LKMS.N 1218-1 —— — — LKMS.R 1218-1 36 μg/cm² 75 3.6 μg/cm² 20 NGSR.N 1218-1 36 μg/cm²77 3.6 μg/cm² 23 50 mM Carbonate — 0 — 0 Buffer (pH10) 49PVD 36 μg/cm² 03.6 μg/cm² 2 LKMS.N49PVD 36 μg/cm² 83 3.6 μg/cm² 0 LKMS.R49PVD 36 μg/cm²62 3.6 μg/cm² 3 NGSR.N49PVD 36 μg/cm² 81 3.6 μg/cm² 25

[0221] TABLE 4 Pesticidal Activity of Cry1218-1 Mutant Polypeptidesagainst Southern Corn Rootworm- Replicate 3 % MOR- % MOR- POLYPEPTIDEDOSE TALITY DOSE TALITY 1218-1A 36 μg/cm² 2 3.6 μg/cm² 0 LKMS.N 1218-1 —— — — LKMS.R 1218-1 36 μg/cm² 74 3.6 μg/cm² 15 NGSR.N 1218-1 36 μg/cm²65 3.6 μg/cm² 17 50 mM — 0 — 0 Carbonate Buffer (pH 10) 49PVD 36 μg/cm²0 3.6 μg/cm² 0 LKMS.N49PVD 36 μg/cm² 70 3.6 μg/cm² 5 LKMS.R49PVD 36μg/cm² 57 3.6 μg/cm² 4 NGSR.N49PVD 36 μg/cm² 81 3.6 μg/cm² 28

[0222] TABLE 5 Pesticidal Activity of Cry1218-1 Mutant Polypeptidesagainst Western Corn Rootworm POLYPEPTIDE DOSE % MORTALITY 1218-1ALKMS.N 1218-1 LKMS.R 1218-1 88 μg/cm² 16 NGSR.N 1218-1 88 μg/cm² 14 50mM Carbonate Buffer — 4 (pH 10) 49PVD 88 μg/cm² LKMS.N49PVD 88 μg/cm² 7LKMS.R49PVD 88 μg/cm² 12 NGSR.N49PVD 88 μg/cm² 10

[0223] TABLE 6 Pesticidal Activity of Cry1218-1 Mutant Polypeptidesagainst Western Corn Rootwoom AVERAGE NUMBER OF LARVAE DOSE LARVAE WT(μg) WEIGHED % MORTALITY POLYPEPTIDE (μg/cm²) TRAY 1 TRAY 2 AVERAGE TRAY1 TRAY 2 AVERAGE TRAY 1 TRAY 2 AVERAGE 1218-1A 193 161 140 150.5 74 6569.5 9 9 9 NGSR.N 193 92 83 99 60 60 60 24 12 18 1218-1 LKMS.R 193 92106 99 48 49 48.5 20 13 16.5 1218-1 49PVD 220 129 166 147.5 79 71 75 6 24 NGSR.N 220 67 76 71.5 39 58 48.5 22 7 14.5 49PVD LKMS.R 220 92 94 9349 32 40.5 20 17 18.5 49PVD LKMS.N 220 82 80 81 44 41 42.5 28 16 2249PVD 50 Mm 220 165 164 164.5 80 95 87.5 4 0 2 Carbonate Diet 220 171132 151.5 78 78 78 1 6 3.5

Example 8 Transformation of Maize by Particle Bombardment andRegeneration of Transgenic Plants

[0224] Immature maize embryos from greenhouse donor plants are bombardedwith a plasmid containing the plant-optimized Cry1218-1 nucleotidesequence (SEQ ID NO:9) operably linked to a ubiquitin promoter and theselectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37),which confers resistance to the herbicide Bialaphos. Alternatively, theselectable marker gene is provided on a separate plasmid. Transformationis performed as follows. Media recipes follow below.

[0225] Preparation of Target Tissue

[0226] The ears are husked and surface sterilized in 30% Clorox bleachplus 0.5% Micro detergent for 20 minutes, and rinsed two times withsterile water. The immature embryos are excised and placed embryo axisside down (scutellum side up), 25 embryos per plate, on 560Y medium for4 hours and then aligned within the 2.5-cm target zone in preparationfor bombardment.

[0227] Preparation of DNA

[0228] A plasmid vector comprising the plant-optimized Cry1218-1nucleotide sequence (SEQ ID NO:9) operably linked to a ubiquitinpromoter is made. For example, a suitable transformation vectorcomprises a UBI1 promoter from Zea mays, a 5′ UTR from UBI1 and a UBI1intron, in combination with a PinII terminator. A plasmid DNA comprisingthe plant-optimized nucleotide sequence and a second plasmid DNAcontaining a PAT selectable marker (e.g., CAMV35S(ACK) promoter drivingPAT with a CAMV35S terminator) is precipitated onto 1.1 μm (averagediameter) tungsten pellets using a CaCl₂ precipitation procedure asfollows:

[0229] 100 μl prepared tungsten particles in water

[0230] 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)

[0231] 100 μl 2.5 M CaCl₂

[0232] 10 μl 0.1 M spermidine

[0233] Each reagent is added sequentially to a tungsten particlesuspension, while maintained on the multitube vortexer. The finalmixture is sonicated briefly and allowed to incubate under constantvortexing for 10 minutes. After the precipitation period, the tubes arecentrifuged briefly, liquid removed, washed with 500 ml 100% ethanol,and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl100% ethanol is added to the final tungsten particle pellet. Forparticle gun bombardment, the tungsten/DNA particles are brieflysonicated and 10 μl spotted onto the center of each macrocarrier andallowed to dry about 2 minutes before bombardment.

[0234] Particle Gun Treatment

[0235] The sample plates are bombarded at level #4 in particle gun#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with atotal of ten aliquots taken from each tube of prepared particles/DNA.

[0236] Subsequent Treatment

[0237] Following bombardment, the embryos are kept on 560Y medium for 2days, then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5′ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for expression of the Cry 1218-1 proteinby assays known in the art, such as, for example, immunoassays andwestern blotting with an antibody that binds to the Cry1218-1 protein.

[0238] Bombardment and Culture Media

[0239] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂0 following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂0); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511),0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought tovolume with D-I H₂0 following adjustment to pH 5.8 with KOH); 3.0 g/lGelrite (added after bringing to volume with D-I H₂0); and 0.85 mg/lsilver nitrate and 3.0 mg/l Bialaphos (both added after sterilizing themedium and cooling to room temperature).

[0240] Plant regeneration medium (288J) comprises 4.3 g/l MS salts(GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 gnicotinic acid, 0.02 g/l thiamine HCl, 0.10 g/l pyridoxine HCl, and 0.40g/l Glycine brought to volume with polished D-I H₂0) (Murashige andSkoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/lzeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought tovolume with polished D-I H₂0 after adjusting to pH 5.6); 3.0 g/l Gelrite(added after bringing to volume with D-I H₂0); and 1.0 mg/l indoleaceticacid and 3.0 mg/l Bialaphos (added after sterilizing the medium andcooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MSsalts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/lnicotinic acid, 0.02 g/l thiamine HCl, 0.10 g/l pyridoxine HCl, and 0.40g/l Glycine brought to volume with polished D-1 H₂0), 0.1 g/lmyo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-IH₂0 after adjusting pH to 5.6); and 6 g/l bacto-agar (added afterbringing to volume with polished D-I H₂0), sterilized and cooled to 60°C.

Example 9 Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

[0241] For Agrobacterium-mediated transformation of maize with aplant-optimized Cry1218-1 nucleotide sequence (SEQ ID NO: 9), preferablythe method of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT patentpublication WO98/32326; the contents of which are hereby incorporated byreference). Briefly, immature embryos are isolated from maize and theembryos contacted with a suspension of Agrobacterium, under conditionswhereby the bacteria are capable of transferring the plant-optimizedCry1218-1 nucleotide sequence (SEQ ID NO:9) to at least one cell of atleast one of the immature embryos (step 1: the infection step). In thisstep the immature embryos are preferably immersed in an Agrobacteriumsuspension for the initiation of inoculation. The embryos areco-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). Preferably the immature embryos are cultured onsolid medium following the infection step. Following this co-cultivationperiod an optional “resting” step is contemplated. In this resting step,the embryos are incubated in the presence of at least one antibioticknown to inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step).Preferably the immature embryos are cultured on solid medium withantibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells. Next,inoculated embryos are cultured on medium containing a selective agentand growing transformed callus is recovered (step 4: the selectionstep). Preferably, the immature embryos are cultured on solid mediumwith a selective agent resulting in the selective growth of transformedcells. The callus is then regenerated into plants (step 5: theregeneration step), and preferably calli grown on selective medium arecultured on solid medium to regenerate the plants.

[0242] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0243] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended embodiments.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 48 <210> SEQ ID NO 1<211> LENGTH: 3621 <212> TYPE: DNA <213> ORGANISM: Bacillusthuringiensis <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)...(3621) <221> NAME/KEY: misc_feature <222> LOCATION: (0)...(0)<223> OTHER INFORMATION: Cry1218-1 <400> SEQUENCE: 1 atg agt cca aat aatcaa aat gaa tat gaa att ata gat gcg aca cct 48 Met Ser Pro Asn Asn GlnAsn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 tct act tct gta tccaat gat tct aac aga tac cct ttt gcg aat gag 96 Ser Thr Ser Val Ser AsnAsp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 cca aca aat gcg cta caaaat atg gat tat aaa gat tat tta aaa atg 144 Pro Thr Asn Ala Leu Gln AsnMet Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45 tct gcg gga aat gct agt gaatac cct ggt tca cct gaa gta ctt gtt 192 Ser Ala Gly Asn Ala Ser Glu TyrPro Gly Ser Pro Glu Val Leu Val 50 55 60 agc gga caa gat gca gct aag gccgca att gat ata gta ggt aaa tta 240 Ser Gly Gln Asp Ala Ala Lys Ala AlaIle Asp Ile Val Gly Lys Leu 65 70 75 80 cta tca ggt tta ggg gtc cca tttgtt ggg ccg ata gtg agt ctt tat 288 Leu Ser Gly Leu Gly Val Pro Phe ValGly Pro Ile Val Ser Leu Tyr 85 90 95 act caa ctt att gat att ctg tgg ccttca ggg gaa aag agt caa tgg 336 Thr Gln Leu Ile Asp Ile Leu Trp Pro SerGly Glu Lys Ser Gln Trp 100 105 110 gaa att ttt atg gaa caa gta gaa gaactc att aat caa aaa ata gca 384 Glu Ile Phe Met Glu Gln Val Glu Glu LeuIle Asn Gln Lys Ile Ala 115 120 125 gaa tat gca agg aat aaa gcg ctt tcggaa tta gaa gga tta ggt aat 432 Glu Tyr Ala Arg Asn Lys Ala Leu Ser GluLeu Glu Gly Leu Gly Asn 130 135 140 aat tac caa tta tat cta act gcg cttgaa gaa tgg gaa gaa aat cca 480 Asn Tyr Gln Leu Tyr Leu Thr Ala Leu GluGlu Trp Glu Glu Asn Pro 145 150 155 160 aat ggt tca aga gcc tta cga gatgtg cga aat cga ttt gaa atc ctg 528 Asn Gly Ser Arg Ala Leu Arg Asp ValArg Asn Arg Phe Glu Ile Leu 165 170 175 gat agt tta ttt acg caa tat atgcca tct ttt aga gtg aca aat ttt 576 Asp Ser Leu Phe Thr Gln Tyr Met ProSer Phe Arg Val Thr Asn Phe 180 185 190 gaa gta cca ttc ctt act gta tatgca atg gca gcc aac ctt cat tta 624 Glu Val Pro Phe Leu Thr Val Tyr AlaMet Ala Ala Asn Leu His Leu 195 200 205 ctg tta tta aag gac gcg tca attttt gga gaa gaa tgg gga tgg tca 672 Leu Leu Leu Lys Asp Ala Ser Ile PheGly Glu Glu Trp Gly Trp Ser 210 215 220 aca act act att aat aac tat tatgat cgt caa atg aaa ctt act gca 720 Thr Thr Thr Ile Asn Asn Tyr Tyr AspArg Gln Met Lys Leu Thr Ala 225 230 235 240 gaa tat tct gat cac tgt gtaaag tgg tat gaa act ggt tta gca aaa 768 Glu Tyr Ser Asp His Cys Val LysTrp Tyr Glu Thr Gly Leu Ala Lys 245 250 255 tta aaa ggc acg agc gct aaacaa tgg gtt gac tat aac caa ttc cgt 816 Leu Lys Gly Thr Ser Ala Lys GlnTrp Val Asp Tyr Asn Gln Phe Arg 260 265 270 aga gaa atg aca ctg gcg gtttta gat gtt gtt gca tta ttc cca aat 864 Arg Glu Met Thr Leu Ala Val LeuAsp Val Val Ala Leu Phe Pro Asn 275 280 285 tat gac aca cgc acg tac ccaatg gaa acg aaa gca caa cta aca agg 912 Tyr Asp Thr Arg Thr Tyr Pro MetGlu Thr Lys Ala Gln Leu Thr Arg 290 295 300 gaa gta tat aca gat cca ctgggc gcg gta aac gtg tct tca att ggt 960 Glu Val Tyr Thr Asp Pro Leu GlyAla Val Asn Val Ser Ser Ile Gly 305 310 315 320 tcc tgg tat gac aaa gcacct tct ttc gga gtg ata gaa tca tcc gtt 1008 Ser Trp Tyr Asp Lys Ala ProSer Phe Gly Val Ile Glu Ser Ser Val 325 330 335 att cga cca ccc cat gtattt gat tat ata acg gga ctc aca gtg tat 1056 Ile Arg Pro Pro His Val PheAsp Tyr Ile Thr Gly Leu Thr Val Tyr 340 345 350 aca caa tca aga agc atttct tcc gct cgc tat ata aga cat tgg gct 1104 Thr Gln Ser Arg Ser Ile SerSer Ala Arg Tyr Ile Arg His Trp Ala 355 360 365 ggt cat caa ata agc taccat cgt gtc agt agg ggt agt aat ctt caa 1152 Gly His Gln Ile Ser Tyr HisArg Val Ser Arg Gly Ser Asn Leu Gln 370 375 380 caa atg tat gga act aatcaa aat cta cac agc act agt acc ttt gat 1200 Gln Met Tyr Gly Thr Asn GlnAsn Leu His Ser Thr Ser Thr Phe Asp 385 390 395 400 ttt acg aat tat gatatt tac aag act cta tca aag gat gca gta ctc 1248 Phe Thr Asn Tyr Asp IleTyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405 410 415 ctt gat att gtt taccct ggt tat acg tat ata ttt ttt gga atg cca 1296 Leu Asp Ile Val Tyr ProGly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420 425 430 gaa gtc gag ttt ttcatg gta aac caa ttg aat aat acc aga aag acg 1344 Glu Val Glu Phe Phe MetVal Asn Gln Leu Asn Asn Thr Arg Lys Thr 435 440 445 tta aag tat aat ccagtt tcc aaa gat att ata gcg agt aca aga gat 1392 Leu Lys Tyr Asn Pro ValSer Lys Asp Ile Ile Ala Ser Thr Arg Asp 450 455 460 tcg gaa tta gaa ttacct cca gaa act tca gat caa cca aat tat gag 1440 Ser Glu Leu Glu Leu ProPro Glu Thr Ser Asp Gln Pro Asn Tyr Glu 465 470 475 480 tca tat agc cataga tta tgt cat atc aca agt att ccc gcg acg ggt 1488 Ser Tyr Ser His ArgLeu Cys His Ile Thr Ser Ile Pro Ala Thr Gly 485 490 495 aac act acc ggatta gta cct gta ttt tct tgg aca cat cga agt gca 1536 Asn Thr Thr Gly LeuVal Pro Val Phe Ser Trp Thr His Arg Ser Ala 500 505 510 gat tta aac aataca ata tat tca gat aaa atc act caa att ccg gcc 1584 Asp Leu Asn Asn ThrIle Tyr Ser Asp Lys Ile Thr Gln Ile Pro Ala 515 520 525 gtt aaa tgt tgggat aat tta ccg ttt gtt cca gtg gta aaa gga cca 1632 Val Lys Cys Trp AspAsn Leu Pro Phe Val Pro Val Val Lys Gly Pro 530 535 540 gga cat aca ggaggg gat tta tta cag tat aat aga agt act ggt tct 1680 Gly His Thr Gly GlyAsp Leu Leu Gln Tyr Asn Arg Ser Thr Gly Ser 545 550 555 560 gta gga acctta ttt cta gct cga tat ggc cta gca tta gaa aaa gca 1728 Val Gly Thr LeuPhe Leu Ala Arg Tyr Gly Leu Ala Leu Glu Lys Ala 565 570 575 ggg aaa tatcgt gta aga ctg aga tat gct act gat gca gat att gta 1776 Gly Lys Tyr ArgVal Arg Leu Arg Tyr Ala Thr Asp Ala Asp Ile Val 580 585 590 ttg cat gtaaac gat gct cag att cag atg cca aaa aca atg aac cca 1824 Leu His Val AsnAsp Ala Gln Ile Gln Met Pro Lys Thr Met Asn Pro 595 600 605 ggt gag gatctg aca tct aaa act ttt aaa gtt gca gat gct atc aca 1872 Gly Glu Asp LeuThr Ser Lys Thr Phe Lys Val Ala Asp Ala Ile Thr 610 615 620 aca tta aattta gca aca gat agt tcg cta gca ttg aaa cat aat tta 1920 Thr Leu Asn LeuAla Thr Asp Ser Ser Leu Ala Leu Lys His Asn Leu 625 630 635 640 ggt gaagac cct aat tca aca tta tct ggt ata gtt tac gtt gac cga 1968 Gly Glu AspPro Asn Ser Thr Leu Ser Gly Ile Val Tyr Val Asp Arg 645 650 655 atc gaattc atc cca gta gat gag aca tat gaa gcg gaa caa gat tta 2016 Ile Glu PheIle Pro Val Asp Glu Thr Tyr Glu Ala Glu Gln Asp Leu 660 665 670 gaa gcagcg aag aaa gca gtg aat gcc ttg ttt acg aat aca aaa gat 2064 Glu Ala AlaLys Lys Ala Val Asn Ala Leu Phe Thr Asn Thr Lys Asp 675 680 685 ggc ttacga cca ggc gta acg gat tat gaa gtg aat caa gcg gca aac 2112 Gly Leu ArgPro Gly Val Thr Asp Tyr Glu Val Asn Gln Ala Ala Asn 690 695 700 tta gtggaa tgc cta tcg gat gat ttg tat cca aat gaa aaa cga ttg 2160 Leu Val GluCys Leu Ser Asp Asp Leu Tyr Pro Asn Glu Lys Arg Leu 705 710 715 720 ttattt gat gca gtg aga gag gca aaa cgc ctc agt gag gca cgt aat 2208 Leu PheAsp Ala Val Arg Glu Ala Lys Arg Leu Ser Glu Ala Arg Asn 725 730 735 ttgctt caa gat cca gat ttc caa gag ata aat gga gaa aat ggc tgg 2256 Leu LeuGln Asp Pro Asp Phe Gln Glu Ile Asn Gly Glu Asn Gly Trp 740 745 750 acggca agt acg gga att gag gtt ata gaa ggg gat gct tta ttc aaa 2304 Thr AlaSer Thr Gly Ile Glu Val Ile Glu Gly Asp Ala Leu Phe Lys 755 760 765 gggcgt tat cta cgc cta cca ggt gcg aga gaa ata gat acg gaa acg 2352 Gly ArgTyr Leu Arg Leu Pro Gly Ala Arg Glu Ile Asp Thr Glu Thr 770 775 780 tatcca acg tat ctg tat caa aaa gta gag gaa ggt gta tta aaa cca 2400 Tyr ProThr Tyr Leu Tyr Gln Lys Val Glu Glu Gly Val Leu Lys Pro 785 790 795 800tac aca aga tat aga ttg aga ggg ttt gtc gga agc agt caa gga ttg 2448 TyrThr Arg Tyr Arg Leu Arg Gly Phe Val Gly Ser Ser Gln Gly Leu 805 810 815gaa att ttc aca att cgt cat caa acg aac cga att gta aaa aat gta 2496 GluIle Phe Thr Ile Arg His Gln Thr Asn Arg Ile Val Lys Asn Val 820 825 830ccg gat gat ttg ctg cca gat gta tct cct gtt aac tcg gat ggt agt 2544 ProAsp Asp Leu Leu Pro Asp Val Ser Pro Val Asn Ser Asp Gly Ser 835 840 845atc aat cga tgc agc gaa caa aag tat gtg aat agc cgt tta gaa gta 2592 IleAsn Arg Cys Ser Glu Gln Lys Tyr Val Asn Ser Arg Leu Glu Val 850 855 860gaa aac cgt tct ggt gaa gcg cat gag ttc tct att cct att gat aca 2640 GluAsn Arg Ser Gly Glu Ala His Glu Phe Ser Ile Pro Ile Asp Thr 865 870 875880 ggt gaa atc gat tac aat gaa aat gca gga ata tgg gtt gga ttt aag 2688Gly Glu Ile Asp Tyr Asn Glu Asn Ala Gly Ile Trp Val Gly Phe Lys 885 890895 att acg gac cca gag gga tat gca aca ctc gga aac cta gaa ttg gtc 2736Ile Thr Asp Pro Glu Gly Tyr Ala Thr Leu Gly Asn Leu Glu Leu Val 900 905910 gaa gag gga cct tta tca gga gac gca tta gaa cgc ttg caa aga gaa 2784Glu Glu Gly Pro Leu Ser Gly Asp Ala Leu Glu Arg Leu Gln Arg Glu 915 920925 gaa caa cag tgg aag att caa atg aca aga aga cgt gaa gaa aca gat 2832Glu Gln Gln Trp Lys Ile Gln Met Thr Arg Arg Arg Glu Glu Thr Asp 930 935940 aga agg tat atg gca tcg aaa caa gcg gta gat cgt tta tat gcc gat 2880Arg Arg Tyr Met Ala Ser Lys Gln Ala Val Asp Arg Leu Tyr Ala Asp 945 950955 960 tat cag gat cag caa ctg aat cct gat gta gag att aca gat ctt act2928 Tyr Gln Asp Gln Gln Leu Asn Pro Asp Val Glu Ile Thr Asp Leu Thr 965970 975 gcg gcc caa gat ctg ata cag tcc att cct tac gta tat aac gaa atg2976 Ala Ala Gln Asp Leu Ile Gln Ser Ile Pro Tyr Val Tyr Asn Glu Met 980985 990 ttc cca gaa ata cca ggg atg aac tat acg aag ttt aca gaa tta aca3024 Phe Pro Glu Ile Pro Gly Met Asn Tyr Thr Lys Phe Thr Glu Leu Thr 9951000 1005 gat cga ctc caa caa gcg tgg agt ttg tat gat cag cga aat gccata 3072 Asp Arg Leu Gln Gln Ala Trp Ser Leu Tyr Asp Gln Arg Asn Ala Ile1010 1015 1020 cca aat ggt gat ttt cga aat ggg tta agt aat tgg aat gcaacg cct 3120 Pro Asn Gly Asp Phe Arg Asn Gly Leu Ser Asn Trp Asn Ala ThrPro 1025 1030 1035 1040 ggc gta gaa gta caa caa atc aat cat aca tct gtcctt gtg att cca 3168 Gly Val Glu Val Gln Gln Ile Asn His Thr Ser Val LeuVal Ile Pro 1045 1050 1055 aac tgg gat gag caa gtt tcg caa cag ttt acagtt caa ccg aat caa 3216 Asn Trp Asp Glu Gln Val Ser Gln Gln Phe Thr ValGln Pro Asn Gln 1060 1065 1070 aga tat gtg tta cga gtt act gcg aga aaagaa ggg gta gga aat gga 3264 Arg Tyr Val Leu Arg Val Thr Ala Arg Lys GluGly Val Gly Asn Gly 1075 1080 1085 tat gta agt atc cgt gat ggt gga aatcaa aca gaa acg ctt act ttt 3312 Tyr Val Ser Ile Arg Asp Gly Gly Asn GlnThr Glu Thr Leu Thr Phe 1090 1095 1100 agt gca agc gat tat gat aca aatgga atg tat aat acg caa gtg tcc 3360 Ser Ala Ser Asp Tyr Asp Thr Asn GlyMet Tyr Asn Thr Gln Val Ser 1105 1110 1115 1120 aat aca aat gga tat aacaca aat aat gcg tat aat aca caa gca tcg 3408 Asn Thr Asn Gly Tyr Asn ThrAsn Asn Ala Tyr Asn Thr Gln Ala Ser 1125 1130 1135 agt aca aac gga tataac gca aat aat atg tat aat acg caa gca tcg 3456 Ser Thr Asn Gly Tyr AsnAla Asn Asn Met Tyr Asn Thr Gln Ala Ser 1140 1145 1150 aat aca aac ggatat aac aca aat agt gtg tac aat gat caa acc ggc 3504 Asn Thr Asn Gly TyrAsn Thr Asn Ser Val Tyr Asn Asp Gln Thr Gly 1155 1160 1165 tat atc acaaaa aca gtg aca ttc atc ccg tat aca gat caa atg tgg 3552 Tyr Ile Thr LysThr Val Thr Phe Ile Pro Tyr Thr Asp Gln Met Trp 1170 1175 1180 att gagatg agt gag aca gaa ggt aca ttc tat ata gaa agt gta gaa 3600 Ile Glu MetSer Glu Thr Glu Gly Thr Phe Tyr Ile Glu Ser Val Glu 1185 1190 1195 1200ttg att gta gac gta gag taa 3621 Leu Ile Val Asp Val Glu * 1205 <210>SEQ ID NO 2 <211> LENGTH: 1206 <212> TYPE: PRT <213> ORGANISM: Bacillusthuringiensis <400> SEQUENCE: 2 Met Ser Pro Asn Asn Gln Asn Glu Tyr GluIle Ile Asp Ala Thr Pro 1 5 10 15 Ser Thr Ser Val Ser Asn Asp Ser AsnArg Tyr Pro Phe Ala Asn Glu 20 25 30 Pro Thr Asn Ala Leu Gln Asn Met AspTyr Lys Asp Tyr Leu Lys Met 35 40 45 Ser Ala Gly Asn Ala Ser Glu Tyr ProGly Ser Pro Glu Val Leu Val 50 55 60 Ser Gly Gln Asp Ala Ala Lys Ala AlaIle Asp Ile Val Gly Lys Leu 65 70 75 80 Leu Ser Gly Leu Gly Val Pro PheVal Gly Pro Ile Val Ser Leu Tyr 85 90 95 Thr Gln Leu Ile Asp Ile Leu TrpPro Ser Gly Glu Lys Ser Gln Trp 100 105 110 Glu Ile Phe Met Glu Gln ValGlu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 Glu Tyr Ala Arg Asn LysAla Leu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 Asn Tyr Gln Leu TyrLeu Thr Ala Leu Glu Glu Trp Glu Glu Asn Pro 145 150 155 160 Asn Gly SerArg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu Ile Leu 165 170 175 Asp SerLeu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn Phe 180 185 190 GluVal Pro Phe Leu Thr Val Tyr Ala Met Ala Ala Asn Leu His Leu 195 200 205Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp Ser 210 215220 Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu Thr Ala 225230 235 240 Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu AlaLys 245 250 255 Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr Asn GlnPhe Arg 260 265 270 Arg Glu Met Thr Leu Ala Val Leu Asp Val Val Ala LeuPhe Pro Asn 275 280 285 Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys AlaGln Leu Thr Arg 290 295 300 Glu Val Tyr Thr Asp Pro Leu Gly Ala Val AsnVal Ser Ser Ile Gly 305 310 315 320 Ser Trp Tyr Asp Lys Ala Pro Ser PheGly Val Ile Glu Ser Ser Val 325 330 335 Ile Arg Pro Pro His Val Phe AspTyr Ile Thr Gly Leu Thr Val Tyr 340 345 350 Thr Gln Ser Arg Ser Ile SerSer Ala Arg Tyr Ile Arg His Trp Ala 355 360 365 Gly His Gln Ile Ser TyrHis Arg Val Ser Arg Gly Ser Asn Leu Gln 370 375 380 Gln Met Tyr Gly ThrAsn Gln Asn Leu His Ser Thr Ser Thr Phe Asp 385 390 395 400 Phe Thr AsnTyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405 410 415 Leu AspIle Val Tyr Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420 425 430 GluVal Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys Thr 435 440 445Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Ser Thr Arg Asp 450 455460 Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr Glu 465470 475 480 Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala ThrGly 485 490 495 Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr His ArgSer Ala 500 505 510 Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile Thr GlnIle Pro Ala 515 520 525 Val Lys Cys Trp Asp Asn Leu Pro Phe Val Pro ValVal Lys Gly Pro 530 535 540 Gly His Thr Gly Gly Asp Leu Leu Gln Tyr AsnArg Ser Thr Gly Ser 545 550 555 560 Val Gly Thr Leu Phe Leu Ala Arg TyrGly Leu Ala Leu Glu Lys Ala 565 570 575 Gly Lys Tyr Arg Val Arg Leu ArgTyr Ala Thr Asp Ala Asp Ile Val 580 585 590 Leu His Val Asn Asp Ala GlnIle Gln Met Pro Lys Thr Met Asn Pro 595 600 605 Gly Glu Asp Leu Thr SerLys Thr Phe Lys Val Ala Asp Ala Ile Thr 610 615 620 Thr Leu Asn Leu AlaThr Asp Ser Ser Leu Ala Leu Lys His Asn Leu 625 630 635 640 Gly Glu AspPro Asn Ser Thr Leu Ser Gly Ile Val Tyr Val Asp Arg 645 650 655 Ile GluPhe Ile Pro Val Asp Glu Thr Tyr Glu Ala Glu Gln Asp Leu 660 665 670 GluAla Ala Lys Lys Ala Val Asn Ala Leu Phe Thr Asn Thr Lys Asp 675 680 685Gly Leu Arg Pro Gly Val Thr Asp Tyr Glu Val Asn Gln Ala Ala Asn 690 695700 Leu Val Glu Cys Leu Ser Asp Asp Leu Tyr Pro Asn Glu Lys Arg Leu 705710 715 720 Leu Phe Asp Ala Val Arg Glu Ala Lys Arg Leu Ser Glu Ala ArgAsn 725 730 735 Leu Leu Gln Asp Pro Asp Phe Gln Glu Ile Asn Gly Glu AsnGly Trp 740 745 750 Thr Ala Ser Thr Gly Ile Glu Val Ile Glu Gly Asp AlaLeu Phe Lys 755 760 765 Gly Arg Tyr Leu Arg Leu Pro Gly Ala Arg Glu IleAsp Thr Glu Thr 770 775 780 Tyr Pro Thr Tyr Leu Tyr Gln Lys Val Glu GluGly Val Leu Lys Pro 785 790 795 800 Tyr Thr Arg Tyr Arg Leu Arg Gly PheVal Gly Ser Ser Gln Gly Leu 805 810 815 Glu Ile Phe Thr Ile Arg His GlnThr Asn Arg Ile Val Lys Asn Val 820 825 830 Pro Asp Asp Leu Leu Pro AspVal Ser Pro Val Asn Ser Asp Gly Ser 835 840 845 Ile Asn Arg Cys Ser GluGln Lys Tyr Val Asn Ser Arg Leu Glu Val 850 855 860 Glu Asn Arg Ser GlyGlu Ala His Glu Phe Ser Ile Pro Ile Asp Thr 865 870 875 880 Gly Glu IleAsp Tyr Asn Glu Asn Ala Gly Ile Trp Val Gly Phe Lys 885 890 895 Ile ThrAsp Pro Glu Gly Tyr Ala Thr Leu Gly Asn Leu Glu Leu Val 900 905 910 GluGlu Gly Pro Leu Ser Gly Asp Ala Leu Glu Arg Leu Gln Arg Glu 915 920 925Glu Gln Gln Trp Lys Ile Gln Met Thr Arg Arg Arg Glu Glu Thr Asp 930 935940 Arg Arg Tyr Met Ala Ser Lys Gln Ala Val Asp Arg Leu Tyr Ala Asp 945950 955 960 Tyr Gln Asp Gln Gln Leu Asn Pro Asp Val Glu Ile Thr Asp LeuThr 965 970 975 Ala Ala Gln Asp Leu Ile Gln Ser Ile Pro Tyr Val Tyr AsnGlu Met 980 985 990 Phe Pro Glu Ile Pro Gly Met Asn Tyr Thr Lys Phe ThrGlu Leu Thr 995 1000 1005 Asp Arg Leu Gln Gln Ala Trp Ser Leu Tyr AspGln Arg Asn Ala Ile 1010 1015 1020 Pro Asn Gly Asp Phe Arg Asn Gly LeuSer Asn Trp Asn Ala Thr Pro 1025 1030 1035 1040 Gly Val Glu Val Gln GlnIle Asn His Thr Ser Val Leu Val Ile Pro 1045 1050 1055 Asn Trp Asp GluGln Val Ser Gln Gln Phe Thr Val Gln Pro Asn Gln 1060 1065 1070 Arg TyrVal Leu Arg Val Thr Ala Arg Lys Glu Gly Val Gly Asn Gly 1075 1080 1085Tyr Val Ser Ile Arg Asp Gly Gly Asn Gln Thr Glu Thr Leu Thr Phe 10901095 1100 Ser Ala Ser Asp Tyr Asp Thr Asn Gly Met Tyr Asn Thr Gln ValSer 1105 1110 1115 1120 Asn Thr Asn Gly Tyr Asn Thr Asn Asn Ala Tyr AsnThr Gln Ala Ser 1125 1130 1135 Ser Thr Asn Gly Tyr Asn Ala Asn Asn MetTyr Asn Thr Gln Ala Ser 1140 1145 1150 Asn Thr Asn Gly Tyr Asn Thr AsnSer Val Tyr Asn Asp Gln Thr Gly 1155 1160 1165 Tyr Ile Thr Lys Thr ValThr Phe Ile Pro Tyr Thr Asp Gln Met Trp 1170 1175 1180 Ile Glu Met SerGlu Thr Glu Gly Thr Phe Tyr Ile Glu Ser Val Glu 1185 1190 1195 1200 LeuIle Val Asp Val Glu 1205 <210> SEQ ID NO 3 <211> LENGTH: 3633 <212>TYPE: DNA <213> ORGANISM: Bacillus thuringiensis <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)...(3633) <221> NAME/KEY: misc_feature<222> LOCATION: (0)...(0) <223> OTHER INFORMATION: Cry1218-2 <400>SEQUENCE: 3 atg agt cca aat aat caa aat gaa tat gaa att ata gat gcg acacct 48 Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 15 10 15 tct act tct gta tcc aat gat tct aac aga tac cct ttt gcg aat gag96 Ser Thr Ser Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 2530 cca aca aat gcg cta caa aat atg gat tat aaa gat tat tta aaa atg 144Pro Thr Asn Ala Leu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45tct gcg gga aat gct agt gaa tac cct ggt tca cct gaa gta ctt gtt 192 SerAla Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 50 55 60 agcgga caa gat gca gct aag gcc gca att gat ata gta ggt aaa tta 240 Ser GlyGln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly Lys Leu 65 70 75 80 ctatca ggt tta ggg gtc cca ttt gtt ggg ccg ata gtg agt ctt tat 288 Leu SerGly Leu Gly Val Pro Phe Val Gly Pro Ile Val Ser Leu Tyr 85 90 95 act caactt att gat att ctg tgg cct tca ggg caa aag agt caa tgg 336 Thr Gln LeuIle Asp Ile Leu Trp Pro Ser Gly Gln Lys Ser Gln Trp 100 105 110 gag attttt atg gaa caa gta gaa gaa ctc ata aat caa aaa ata gca 384 Glu Ile PheMet Glu Gln Val Glu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 gaa tatgca agg aat aaa gcg ctt tcg gaa tta gaa gga tta ggt aat 432 Glu Tyr AlaArg Asn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 aat taccaa tta tat cta act gcg ctt gaa gaa tgg aaa gaa aat cca 480 Asn Tyr GlnLeu Tyr Leu Thr Ala Leu Glu Glu Trp Lys Glu Asn Pro 145 150 155 160 aatggt tca aga gcc tta cga gat gtg cga aat cga ttt gaa atc ctg 528 Asn GlySer Arg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu Ile Leu 165 170 175 gatagt tta ttt acg caa tac atg cca tct ttt cga gtg aca aat ttt 576 Asp SerLeu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn Phe 180 185 190 gaagta cca ttc ctt aca gta tat aca cag gca gcc aac ctt cat tta 624 Glu ValPro Phe Leu Thr Val Tyr Thr Gln Ala Ala Asn Leu His Leu 195 200 205 ctgtta tta aag gac gct tca att ttt gga gaa gaa tgg gga tgg tct 672 Leu LeuLeu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp Ser 210 215 220 acaacc act att aat aac tat tat gat cgt caa atg aaa ctt act gca 720 Thr ThrThr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu Thr Ala 225 230 235 240gaa tat tct gat cac tgt gta aag tgg tat gaa act ggt tta gca aaa 768 GluTyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu Ala Lys 245 250 255tta aaa ggc acg agc gct aaa caa tgg gtc gac tat aac caa ttc cgt 816 LeuLys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr Asn Gln Phe Arg 260 265 270aga gaa atg aca ctg acg gtt tta gat gtt gtt gca tta ttc cca aat 864 ArgGlu Met Thr Leu Thr Val Leu Asp Val Val Ala Leu Phe Pro Asn 275 280 285tat gac aca cgc acg tac cca atg gaa acg aaa gca caa cta aca agg 912 TyrAsp Thr Arg Thr Tyr Pro Met Glu Thr Lys Ala Gln Leu Thr Arg 290 295 300gaa gta tat aca gat cca ctg ggc gcg gta aac gtg tct tca att ggt 960 GluVal Tyr Thr Asp Pro Leu Gly Ala Val Asn Val Ser Ser Ile Gly 305 310 315320 tcc tgg tat gac aaa gca cct tct ttc gga gtg ata gaa tca tcc gtt 1008Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val Ile Glu Ser Ser Val 325 330335 att cga cca ccc cat gta ttt gat tat ata acg gga ctc aca gtg tat 1056Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr Gly Leu Thr Val Tyr 340 345350 aca caa tca aga agc att tct tcc gct cgc tat ata aga cat tgg gct 1104Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr Ile Arg His Trp Ala 355 360365 ggt cat caa ata agc tat cat cgg att ttt agt gat aat att ata aaa 1152Gly His Gln Ile Ser Tyr His Arg Ile Phe Ser Asp Asn Ile Ile Lys 370 375380 cag atg tat gga act aat caa aat cta cac agc act agt acc ttt gat 1200Gln Met Tyr Gly Thr Asn Gln Asn Leu His Ser Thr Ser Thr Phe Asp 385 390395 400 ttt acg aat tat gat att tac aag acg tta tca aaa gat gcg gtg ctc1248 Phe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405410 415 ctt gat att gtt ttt cct ggt tat acg tat ata ttt ttt gga atg cca1296 Leu Asp Ile Val Phe Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420425 430 gaa gtc gag ttt ttc atg gta aac caa ttg aat aat acc aga aag acg1344 Glu Val Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys Thr 435440 445 tta aag tat aat ccg gtt tcc aaa gat att ata gcg ggg aca aga gat1392 Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Gly Thr Arg Asp 450455 460 tcg gaa tta gaa tta cct cca gaa act tca gat caa cca aat tat gag1440 Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr Glu 465470 475 480 tca tat agc cat aga tta tgt cat atc aca agt att ccc gcg acgggt 1488 Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala Thr Gly485 490 495 tca act acc gga tta gta cct gta ttt tct tgg aca cat cgg agtgcc 1536 Ser Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr His Arg Ser Ala500 505 510 gat ctt ata aat gca gtt cat tca gat aaa att act cag att ccggtc 1584 Asp Leu Ile Asn Ala Val His Ser Asp Lys Ile Thr Gln Ile Pro Val515 520 525 gta aag gtt tct gat ttg gct ccc tct ata aca gga ggg cca aataat 1632 Val Lys Val Ser Asp Leu Ala Pro Ser Ile Thr Gly Gly Pro Asn Asn530 535 540 acc gtt gta tcg ggt cct gga ttt aca ggg ggg ggg ata ata aaagta 1680 Thr Val Val Ser Gly Pro Gly Phe Thr Gly Gly Gly Ile Ile Lys Val545 550 555 560 ata aga aat gga gta att ata tca cat atg cgt gtt aaa atttca gac 1728 Ile Arg Asn Gly Val Ile Ile Ser His Met Arg Val Lys Ile SerAsp 565 570 575 att aac aaa gaa tat agt atg agg att cgg tat gct tcc gctaat aat 1776 Ile Asn Lys Glu Tyr Ser Met Arg Ile Arg Tyr Ala Ser Ala AsnAsn 580 585 590 act gaa ttt tat ata aat cct tct gaa gaa aac gtt aaa tctcac gct 1824 Thr Glu Phe Tyr Ile Asn Pro Ser Glu Glu Asn Val Lys Ser HisAla 595 600 605 caa aaa act atg aat aga ggt gaa gct tta aca tat aat aaattt aat 1872 Gln Lys Thr Met Asn Arg Gly Glu Ala Leu Thr Tyr Asn Lys PheAsn 610 615 620 tat gcg act ttg ccc cct att aaa ttt acg aca acc gaa cctttc att 1920 Tyr Ala Thr Leu Pro Pro Ile Lys Phe Thr Thr Thr Glu Pro PheIle 625 630 635 640 act cta ggg gct ata ttt gaa gcg gaa gac ttt ctt ggaatt gaa gct 1968 Thr Leu Gly Ala Ile Phe Glu Ala Glu Asp Phe Leu Gly IleGlu Ala 645 650 655 tat ata gac cga atc gaa ttt atc cca gta gat gag acatat gaa gcg 2016 Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Asp Glu Thr TyrGlu Ala 660 665 670 gaa caa gat tta gaa gca gcg aag aaa gca gtg aat gccttg ttt acg 2064 Glu Gln Asp Leu Glu Ala Ala Lys Lys Ala Val Asn Ala LeuPhe Thr 675 680 685 aat aca aaa gat ggc tta cga cca ggc gta acg gat tatgaa gtg aat 2112 Asn Thr Lys Asp Gly Leu Arg Pro Gly Val Thr Asp Tyr GluVal Asn 690 695 700 caa gcg gca aac tta gtg gaa tgc cta tcg gat gat ttgtat cca aat 2160 Gln Ala Ala Asn Leu Val Glu Cys Leu Ser Asp Asp Leu TyrPro Asn 705 710 715 720 gaa aaa cga ttg tta ttt gat gca gtg aga gag gcaaaa cgc ctc agt 2208 Glu Lys Arg Leu Leu Phe Asp Ala Val Arg Glu Ala LysArg Leu Ser 725 730 735 gag gca cgt aat ttg ctt caa gat cca gat ttc caagag ata aat gga 2256 Glu Ala Arg Asn Leu Leu Gln Asp Pro Asp Phe Gln GluIle Asn Gly 740 745 750 gaa aat ggc tgg acg gca agt acg gga att gag gttata gaa ggg gat 2304 Glu Asn Gly Trp Thr Ala Ser Thr Gly Ile Glu Val IleGlu Gly Asp 755 760 765 gct tta ttc aaa ggg cgt tat cta cgc cta cca ggtgcg aga gaa ata 2352 Ala Leu Phe Lys Gly Arg Tyr Leu Arg Leu Pro Gly AlaArg Glu Ile 770 775 780 gat acg gaa acg tat cca acg tat ctg tat caa aaagta gag gaa ggt 2400 Asp Thr Glu Thr Tyr Pro Thr Tyr Leu Tyr Gln Lys ValGlu Glu Gly 785 790 795 800 gta tta aaa cca tac aca aga tat aga ttg agaggg ttt gtc gga agc 2448 Val Leu Lys Pro Tyr Thr Arg Tyr Arg Leu Arg GlyPhe Val Gly Ser 805 810 815 agt caa gga ttg gaa att ttc aca att cgt catcaa acg aac cga att 2496 Ser Gln Gly Leu Glu Ile Phe Thr Ile Arg His GlnThr Asn Arg Ile 820 825 830 gta aaa aat gta ccg gat gat ttg ctg cca gatgta tct cct gtt aac 2544 Val Lys Asn Val Pro Asp Asp Leu Leu Pro Asp ValSer Pro Val Asn 835 840 845 tcg gat ggt agt atc aat cga tgc agc gaa caaaag tat gtg aat agc 2592 Ser Asp Gly Ser Ile Asn Arg Cys Ser Glu Gln LysTyr Val Asn Ser 850 855 860 cgt tta gaa gta gaa aac cgt tct ggt gaa gcgcat gag ttc tct att 2640 Arg Leu Glu Val Glu Asn Arg Ser Gly Glu Ala HisGlu Phe Ser Ile 865 870 875 880 cct att gat aca ggt gaa atc gat tac aatgaa aat gca gga ata tgg 2688 Pro Ile Asp Thr Gly Glu Ile Asp Tyr Asn GluAsn Ala Gly Ile Trp 885 890 895 gtt gga ttt aag att acg gac cca gag ggatat gca aca ctc gga aac 2736 Val Gly Phe Lys Ile Thr Asp Pro Glu Gly TyrAla Thr Leu Gly Asn 900 905 910 cta gaa ttg gtc gaa gag gga cct tta tcagga gac gca tta gaa cgc 2784 Leu Glu Leu Val Glu Glu Gly Pro Leu Ser GlyAsp Ala Leu Glu Arg 915 920 925 ttg caa aga gaa gaa caa cag tgg aag attcaa atg aca aga aga cgt 2832 Leu Gln Arg Glu Glu Gln Gln Trp Lys Ile GlnMet Thr Arg Arg Arg 930 935 940 gaa gaa aca gat aga agg tat atg gca tcgaaa caa gcg gta gat cgt 2880 Glu Glu Thr Asp Arg Arg Tyr Met Ala Ser LysGln Ala Val Asp Arg 945 950 955 960 tta tat gcc gat tat cag gat cag caactg aat cct gat gta gag att 2928 Leu Tyr Ala Asp Tyr Gln Asp Gln Gln LeuAsn Pro Asp Val Glu Ile 965 970 975 aca gat ctt act gcg gcc caa gat ctgata cag tcc att cct tac gta 2976 Thr Asp Leu Thr Ala Ala Gln Asp Leu IleGln Ser Ile Pro Tyr Val 980 985 990 tat aac gaa atg ttc cca gaa ata ccaggg atg aac tat acg aag ttt 3024 Tyr Asn Glu Met Phe Pro Glu Ile Pro GlyMet Asn Tyr Thr Lys Phe 995 1000 1005 aca gaa tta aca gat cga ctc caacaa gcg tgg agt ttg tat gat cag 3072 Thr Glu Leu Thr Asp Arg Leu Gln GlnAla Trp Ser Leu Tyr Asp Gln 1010 1015 1020 cga aat gcc ata cca aat ggtgat ttt cga aat ggg tta agt aat tgg 3120 Arg Asn Ala Ile Pro Asn Gly AspPhe Arg Asn Gly Leu Ser Asn Trp 1025 1030 1035 1040 aat gca acg cct ggcgta gaa gta caa caa atc aat cat aca tct gtc 3168 Asn Ala Thr Pro Gly ValGlu Val Gln Gln Ile Asn His Thr Ser Val 1045 1050 1055 ctt gtg att ccaaac tgg gat gag caa gtt tcg caa cag ttt aca gtt 3216 Leu Val Ile Pro AsnTrp Asp Glu Gln Val Ser Gln Gln Phe Thr Val 1060 1065 1070 caa ccg aatcaa aga tat gtg tta cga gtt act gcg aga aaa gaa ggg 3264 Gln Pro Asn GlnArg Tyr Val Leu Arg Val Thr Ala Arg Lys Glu Gly 1075 1080 1085 gta ggaaat gga tat gta agt atc cgt gat ggt gga aat caa aca gaa 3312 Val Gly AsnGly Tyr Val Ser Ile Arg Asp Gly Gly Asn Gln Thr Glu 1090 1095 1100 acgctt act ttt agt gca agc gat tat gat aca aat gga atg tat aat 3360 Thr LeuThr Phe Ser Ala Ser Asp Tyr Asp Thr Asn Gly Met Tyr Asn 1105 1110 11151120 acg caa gtg tcc aat aca aat gga tat aac aca aat aat gcg tat aat3408 Thr Gln Val Ser Asn Thr Asn Gly Tyr Asn Thr Asn Asn Ala Tyr Asn1125 1130 1135 aca caa gca tcg agt aca aac gga tat aac gca aat aat atgtat aat 3456 Thr Gln Ala Ser Ser Thr Asn Gly Tyr Asn Ala Asn Asn Met TyrAsn 1140 1145 1150 acg caa gca tcg aat aca aac gga tat aac aca aat agtgtg tac aat 3504 Thr Gln Ala Ser Asn Thr Asn Gly Tyr Asn Thr Asn Ser ValTyr Asn 1155 1160 1165 gat caa acc ggc tat atc aca aaa aca gtg aca ttcatc ccg tat aca 3552 Asp Gln Thr Gly Tyr Ile Thr Lys Thr Val Thr Phe IlePro Tyr Thr 1170 1175 1180 gat caa atg tgg att gag atg agt gag aca gaaggt aca ttc tat ata 3600 Asp Gln Met Trp Ile Glu Met Ser Glu Thr Glu GlyThr Phe Tyr Ile 1185 1190 1195 1200 gaa agt gta gaa ttg att gta gac gtagag taa 3633 Glu Ser Val Glu Leu Ile Val Asp Val Glu * 1205 1210 <210>SEQ ID NO 4 <211> LENGTH: 1210 <212> TYPE: PRT <213> ORGANISM: Bacillusthuringiensis <400> SEQUENCE: 4 Met Ser Pro Asn Asn Gln Asn Glu Tyr GluIle Ile Asp Ala Thr Pro 1 5 10 15 Ser Thr Ser Val Ser Asn Asp Ser AsnArg Tyr Pro Phe Ala Asn Glu 20 25 30 Pro Thr Asn Ala Leu Gln Asn Met AspTyr Lys Asp Tyr Leu Lys Met 35 40 45 Ser Ala Gly Asn Ala Ser Glu Tyr ProGly Ser Pro Glu Val Leu Val 50 55 60 Ser Gly Gln Asp Ala Ala Lys Ala AlaIle Asp Ile Val Gly Lys Leu 65 70 75 80 Leu Ser Gly Leu Gly Val Pro PheVal Gly Pro Ile Val Ser Leu Tyr 85 90 95 Thr Gln Leu Ile Asp Ile Leu TrpPro Ser Gly Gln Lys Ser Gln Trp 100 105 110 Glu Ile Phe Met Glu Gln ValGlu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 Glu Tyr Ala Arg Asn LysAla Leu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 Asn Tyr Gln Leu TyrLeu Thr Ala Leu Glu Glu Trp Lys Glu Asn Pro 145 150 155 160 Asn Gly SerArg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu Ile Leu 165 170 175 Asp SerLeu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn Phe 180 185 190 GluVal Pro Phe Leu Thr Val Tyr Thr Gln Ala Ala Asn Leu His Leu 195 200 205Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp Ser 210 215220 Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu Thr Ala 225230 235 240 Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu AlaLys 245 250 255 Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr Asn GlnPhe Arg 260 265 270 Arg Glu Met Thr Leu Thr Val Leu Asp Val Val Ala LeuPhe Pro Asn 275 280 285 Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys AlaGln Leu Thr Arg 290 295 300 Glu Val Tyr Thr Asp Pro Leu Gly Ala Val AsnVal Ser Ser Ile Gly 305 310 315 320 Ser Trp Tyr Asp Lys Ala Pro Ser PheGly Val Ile Glu Ser Ser Val 325 330 335 Ile Arg Pro Pro His Val Phe AspTyr Ile Thr Gly Leu Thr Val Tyr 340 345 350 Thr Gln Ser Arg Ser Ile SerSer Ala Arg Tyr Ile Arg His Trp Ala 355 360 365 Gly His Gln Ile Ser TyrHis Arg Ile Phe Ser Asp Asn Ile Ile Lys 370 375 380 Gln Met Tyr Gly ThrAsn Gln Asn Leu His Ser Thr Ser Thr Phe Asp 385 390 395 400 Phe Thr AsnTyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405 410 415 Leu AspIle Val Phe Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420 425 430 GluVal Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys Thr 435 440 445Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Gly Thr Arg Asp 450 455460 Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr Glu 465470 475 480 Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala ThrGly 485 490 495 Ser Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr His ArgSer Ala 500 505 510 Asp Leu Ile Asn Ala Val His Ser Asp Lys Ile Thr GlnIle Pro Val 515 520 525 Val Lys Val Ser Asp Leu Ala Pro Ser Ile Thr GlyGly Pro Asn Asn 530 535 540 Thr Val Val Ser Gly Pro Gly Phe Thr Gly GlyGly Ile Ile Lys Val 545 550 555 560 Ile Arg Asn Gly Val Ile Ile Ser HisMet Arg Val Lys Ile Ser Asp 565 570 575 Ile Asn Lys Glu Tyr Ser Met ArgIle Arg Tyr Ala Ser Ala Asn Asn 580 585 590 Thr Glu Phe Tyr Ile Asn ProSer Glu Glu Asn Val Lys Ser His Ala 595 600 605 Gln Lys Thr Met Asn ArgGly Glu Ala Leu Thr Tyr Asn Lys Phe Asn 610 615 620 Tyr Ala Thr Leu ProPro Ile Lys Phe Thr Thr Thr Glu Pro Phe Ile 625 630 635 640 Thr Leu GlyAla Ile Phe Glu Ala Glu Asp Phe Leu Gly Ile Glu Ala 645 650 655 Tyr IleAsp Arg Ile Glu Phe Ile Pro Val Asp Glu Thr Tyr Glu Ala 660 665 670 GluGln Asp Leu Glu Ala Ala Lys Lys Ala Val Asn Ala Leu Phe Thr 675 680 685Asn Thr Lys Asp Gly Leu Arg Pro Gly Val Thr Asp Tyr Glu Val Asn 690 695700 Gln Ala Ala Asn Leu Val Glu Cys Leu Ser Asp Asp Leu Tyr Pro Asn 705710 715 720 Glu Lys Arg Leu Leu Phe Asp Ala Val Arg Glu Ala Lys Arg LeuSer 725 730 735 Glu Ala Arg Asn Leu Leu Gln Asp Pro Asp Phe Gln Glu IleAsn Gly 740 745 750 Glu Asn Gly Trp Thr Ala Ser Thr Gly Ile Glu Val IleGlu Gly Asp 755 760 765 Ala Leu Phe Lys Gly Arg Tyr Leu Arg Leu Pro GlyAla Arg Glu Ile 770 775 780 Asp Thr Glu Thr Tyr Pro Thr Tyr Leu Tyr GlnLys Val Glu Glu Gly 785 790 795 800 Val Leu Lys Pro Tyr Thr Arg Tyr ArgLeu Arg Gly Phe Val Gly Ser 805 810 815 Ser Gln Gly Leu Glu Ile Phe ThrIle Arg His Gln Thr Asn Arg Ile 820 825 830 Val Lys Asn Val Pro Asp AspLeu Leu Pro Asp Val Ser Pro Val Asn 835 840 845 Ser Asp Gly Ser Ile AsnArg Cys Ser Glu Gln Lys Tyr Val Asn Ser 850 855 860 Arg Leu Glu Val GluAsn Arg Ser Gly Glu Ala His Glu Phe Ser Ile 865 870 875 880 Pro Ile AspThr Gly Glu Ile Asp Tyr Asn Glu Asn Ala Gly Ile Trp 885 890 895 Val GlyPhe Lys Ile Thr Asp Pro Glu Gly Tyr Ala Thr Leu Gly Asn 900 905 910 LeuGlu Leu Val Glu Glu Gly Pro Leu Ser Gly Asp Ala Leu Glu Arg 915 920 925Leu Gln Arg Glu Glu Gln Gln Trp Lys Ile Gln Met Thr Arg Arg Arg 930 935940 Glu Glu Thr Asp Arg Arg Tyr Met Ala Ser Lys Gln Ala Val Asp Arg 945950 955 960 Leu Tyr Ala Asp Tyr Gln Asp Gln Gln Leu Asn Pro Asp Val GluIle 965 970 975 Thr Asp Leu Thr Ala Ala Gln Asp Leu Ile Gln Ser Ile ProTyr Val 980 985 990 Tyr Asn Glu Met Phe Pro Glu Ile Pro Gly Met Asn TyrThr Lys Phe 995 1000 1005 Thr Glu Leu Thr Asp Arg Leu Gln Gln Ala TrpSer Leu Tyr Asp Gln 1010 1015 1020 Arg Asn Ala Ile Pro Asn Gly Asp PheArg Asn Gly Leu Ser Asn Trp 1025 1030 1035 1040 Asn Ala Thr Pro Gly ValGlu Val Gln Gln Ile Asn His Thr Ser Val 1045 1050 1055 Leu Val Ile ProAsn Trp Asp Glu Gln Val Ser Gln Gln Phe Thr Val 1060 1065 1070 Gln ProAsn Gln Arg Tyr Val Leu Arg Val Thr Ala Arg Lys Glu Gly 1075 1080 1085Val Gly Asn Gly Tyr Val Ser Ile Arg Asp Gly Gly Asn Gln Thr Glu 10901095 1100 Thr Leu Thr Phe Ser Ala Ser Asp Tyr Asp Thr Asn Gly Met TyrAsn 1105 1110 1115 1120 Thr Gln Val Ser Asn Thr Asn Gly Tyr Asn Thr AsnAsn Ala Tyr Asn 1125 1130 1135 Thr Gln Ala Ser Ser Thr Asn Gly Tyr AsnAla Asn Asn Met Tyr Asn 1140 1145 1150 Thr Gln Ala Ser Asn Thr Asn GlyTyr Asn Thr Asn Ser Val Tyr Asn 1155 1160 1165 Asp Gln Thr Gly Tyr IleThr Lys Thr Val Thr Phe Ile Pro Tyr Thr 1170 1175 1180 Asp Gln Met TrpIle Glu Met Ser Glu Thr Glu Gly Thr Phe Tyr Ile 1185 1190 1195 1200 GluSer Val Glu Leu Ile Val Asp Val Glu 1205 1210 <210> SEQ ID NO 5 <211>LENGTH: 2003 <212> TYPE: DNA <213> ORGANISM: Bacillus thuringiensis(truncated) <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)...(2001) <221> NAME/KEY: misc_feature <222> LOCATION: (0)...(0)<223> OTHER INFORMATION: 1218-1 <400> SEQUENCE: 5 atg agt cca aat aatcaa aat gaa tat gaa att ata gat gcg aca cct 48 Met Ser Pro Asn Asn GlnAsn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 tct act tct gta tccaat gat tct aac aga tac cct ttt gcg aat gag 96 Ser Thr Ser Val Ser AsnAsp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 cca aca aat gcg cta caaaat atg gat tat aaa gat tat tta aaa atg 144 Pro Thr Asn Ala Leu Gln AsnMet Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45 tct gcg gga aat gct agt gaatac cct ggt tca cct gaa gta ctt gtt 192 Ser Ala Gly Asn Ala Ser Glu TyrPro Gly Ser Pro Glu Val Leu Val 50 55 60 agc gga caa gat gca gct aag gccgca att gat ata gta ggt aaa tta 240 Ser Gly Gln Asp Ala Ala Lys Ala AlaIle Asp Ile Val Gly Lys Leu 65 70 75 80 cta tca ggt tta ggg gtc cca tttgtt ggg ccg ata gtg agt ctt tat 288 Leu Ser Gly Leu Gly Val Pro Phe ValGly Pro Ile Val Ser Leu Tyr 85 90 95 act caa ctt att gat att ctg tgg ccttca ggg gaa aag agt caa tgg 336 Thr Gln Leu Ile Asp Ile Leu Trp Pro SerGly Glu Lys Ser Gln Trp 100 105 110 gaa att ttt atg gaa caa gta gaa gaactc att aat caa aaa ata gca 384 Glu Ile Phe Met Glu Gln Val Glu Glu LeuIle Asn Gln Lys Ile Ala 115 120 125 gaa tat gca agg aat aaa gcg ctt tcggaa tta gaa gga tta ggt aat 432 Glu Tyr Ala Arg Asn Lys Ala Leu Ser GluLeu Glu Gly Leu Gly Asn 130 135 140 aat tac caa tta tat cta act gcg cttgaa gaa tgg gaa gaa aat cca 480 Asn Tyr Gln Leu Tyr Leu Thr Ala Leu GluGlu Trp Glu Glu Asn Pro 145 150 155 160 aat ggt tca aga gcc tta cga gatgtg cga aat cga ttt gaa atc ctg 528 Asn Gly Ser Arg Ala Leu Arg Asp ValArg Asn Arg Phe Glu Ile Leu 165 170 175 gat agt tta ttt acg caa tat atgcca tct ttt aga gtg aca aat ttt 576 Asp Ser Leu Phe Thr Gln Tyr Met ProSer Phe Arg Val Thr Asn Phe 180 185 190 gaa gta cca ttc ctt act gta tatgca atg gca gcc aac ctt cat tta 624 Glu Val Pro Phe Leu Thr Val Tyr AlaMet Ala Ala Asn Leu His Leu 195 200 205 ctg tta tta aag gac gcg tca attttt gga gaa gaa tgg gga tgg tca 672 Leu Leu Leu Lys Asp Ala Ser Ile PheGly Glu Glu Trp Gly Trp Ser 210 215 220 aca act act att aat aac tat tatgat cgt caa atg aaa ctt act gca 720 Thr Thr Thr Ile Asn Asn Tyr Tyr AspArg Gln Met Lys Leu Thr Ala 225 230 235 240 gaa tat tct gat cac tgt gtaaag tgg tat gaa act ggt tta gca aaa 768 Glu Tyr Ser Asp His Cys Val LysTrp Tyr Glu Thr Gly Leu Ala Lys 245 250 255 tta aaa ggc acg agc gct aaacaa tgg gtt gac tat aac caa ttc cgt 816 Leu Lys Gly Thr Ser Ala Lys GlnTrp Val Asp Tyr Asn Gln Phe Arg 260 265 270 aga gaa atg aca ctg gcg gtttta gat gtt gtt gca tta ttc cca aat 864 Arg Glu Met Thr Leu Ala Val LeuAsp Val Val Ala Leu Phe Pro Asn 275 280 285 tat gac aca cgc acg tac ccaatg gaa acg aaa gca caa cta aca agg 912 Tyr Asp Thr Arg Thr Tyr Pro MetGlu Thr Lys Ala Gln Leu Thr Arg 290 295 300 gaa gta tat aca gat cca ctgggc gcg gta aac gtg tct tca att ggt 960 Glu Val Tyr Thr Asp Pro Leu GlyAla Val Asn Val Ser Ser Ile Gly 305 310 315 320 tcc tgg tat gac aaa gcacct tct ttc gga gtg ata gaa tca tcc gtt 1008 Ser Trp Tyr Asp Lys Ala ProSer Phe Gly Val Ile Glu Ser Ser Val 325 330 335 att cga cca ccc cat gtattt gat tat ata acg gga ctc aca gtg tat 1056 Ile Arg Pro Pro His Val PheAsp Tyr Ile Thr Gly Leu Thr Val Tyr 340 345 350 aca caa tca aga agc atttct tcc gct cgc tat ata aga cat tgg gct 1104 Thr Gln Ser Arg Ser Ile SerSer Ala Arg Tyr Ile Arg His Trp Ala 355 360 365 ggt cat caa ata agc taccat cgt gtc agt agg ggt agt aat ctt caa 1152 Gly His Gln Ile Ser Tyr HisArg Val Ser Arg Gly Ser Asn Leu Gln 370 375 380 caa atg tat gga act aatcaa aat cta cac agc act agt acc ttt gat 1200 Gln Met Tyr Gly Thr Asn GlnAsn Leu His Ser Thr Ser Thr Phe Asp 385 390 395 400 ttt acg aat tat gatatt tac aag act cta tca aag gat gca gta ctc 1248 Phe Thr Asn Tyr Asp IleTyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405 410 415 ctt gat att gtt taccct ggt tat acg tat ata ttt ttt gga atg cca 1296 Leu Asp Ile Val Tyr ProGly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420 425 430 gaa gtc gag ttt ttcatg gta aac caa ttg aat aat acc aga aag acg 1344 Glu Val Glu Phe Phe MetVal Asn Gln Leu Asn Asn Thr Arg Lys Thr 435 440 445 tta aag tat aat ccagtt tcc aaa gat att ata gcg agt aca aga gat 1392 Leu Lys Tyr Asn Pro ValSer Lys Asp Ile Ile Ala Ser Thr Arg Asp 450 455 460 tcg gaa tta gaa ttacct cca gaa act tca gat caa cca aat tat gag 1440 Ser Glu Leu Glu Leu ProPro Glu Thr Ser Asp Gln Pro Asn Tyr Glu 465 470 475 480 tca tat agc cataga tta tgt cat atc aca agt att ccc gcg acg ggt 1488 Ser Tyr Ser His ArgLeu Cys His Ile Thr Ser Ile Pro Ala Thr Gly 485 490 495 aac act acc ggatta gta cct gta ttt tct tgg aca cat cga agt gca 1536 Asn Thr Thr Gly LeuVal Pro Val Phe Ser Trp Thr His Arg Ser Ala 500 505 510 gat tta aac aataca ata tat tca gat aaa atc act caa att ccg gcc 1584 Asp Leu Asn Asn ThrIle Tyr Ser Asp Lys Ile Thr Gln Ile Pro Ala 515 520 525 gtt aaa tgt tgggat aat tta ccg ttt gtt cca gtg gta aaa gga cca 1632 Val Lys Cys Trp AspAsn Leu Pro Phe Val Pro Val Val Lys Gly Pro 530 535 540 gga cat aca ggaggg gat tta tta cag tat aat aga agt act ggt tct 1680 Gly His Thr Gly GlyAsp Leu Leu Gln Tyr Asn Arg Ser Thr Gly Ser 545 550 555 560 gta gga acctta ttt cta gct cga tat ggc cta gca tta gaa aaa gca 1728 Val Gly Thr LeuPhe Leu Ala Arg Tyr Gly Leu Ala Leu Glu Lys Ala 565 570 575 ggg aaa tatcgt gta aga ctg aga tat gct act gat gca gat att gta 1776 Gly Lys Tyr ArgVal Arg Leu Arg Tyr Ala Thr Asp Ala Asp Ile Val 580 585 590 ttg cat gtaaac gat gct cag att cag atg cca aaa aca atg aac cca 1824 Leu His Val AsnAsp Ala Gln Ile Gln Met Pro Lys Thr Met Asn Pro 595 600 605 ggt gag gatctg aca tct aaa act ttt aaa gtt gca gat gct atc aca 1872 Gly Glu Asp LeuThr Ser Lys Thr Phe Lys Val Ala Asp Ala Ile Thr 610 615 620 aca tta aattta gca aca gat agt tcg cta gca ttg aaa cat aat tta 1920 Thr Leu Asn LeuAla Thr Asp Ser Ser Leu Ala Leu Lys His Asn Leu 625 630 635 640 ggt gaagac cct aat tca aca tta tct ggt ata gtt tac gtt gac cga 1968 Gly Glu AspPro Asn Ser Thr Leu Ser Gly Ile Val Tyr Val Asp Arg 645 650 655 atc gaattc atc cca gta gat gag aca tat gaa gc 2003 Ile Glu Phe Ile Pro Val AspGlu Thr Tyr Glu 660 665 <210> SEQ ID NO 6 <211> LENGTH: 667 <212> TYPE:PRT <213> ORGANISM: Bacillus thuringiensis (truncated) <400> SEQUENCE: 6Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 1 5 1015 Ser Thr Ser Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 2530 Pro Thr Asn Ala Leu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 35 4045 Ser Ala Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 50 5560 Ser Gly Gln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly Lys Leu 65 7075 80 Leu Ser Gly Leu Gly Val Pro Phe Val Gly Pro Ile Val Ser Leu Tyr 8590 95 Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly Glu Lys Ser Gln Trp100 105 110 Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile Asn Gln Lys IleAla 115 120 125 Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu Glu Gly LeuGly Asn 130 135 140 Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu Trp GluGlu Asn Pro 145 150 155 160 Asn Gly Ser Arg Ala Leu Arg Asp Val Arg AsnArg Phe Glu Ile Leu 165 170 175 Asp Ser Leu Phe Thr Gln Tyr Met Pro SerPhe Arg Val Thr Asn Phe 180 185 190 Glu Val Pro Phe Leu Thr Val Tyr AlaMet Ala Ala Asn Leu His Leu 195 200 205 Leu Leu Leu Lys Asp Ala Ser IlePhe Gly Glu Glu Trp Gly Trp Ser 210 215 220 Thr Thr Thr Ile Asn Asn TyrTyr Asp Arg Gln Met Lys Leu Thr Ala 225 230 235 240 Glu Tyr Ser Asp HisCys Val Lys Trp Tyr Glu Thr Gly Leu Ala Lys 245 250 255 Leu Lys Gly ThrSer Ala Lys Gln Trp Val Asp Tyr Asn Gln Phe Arg 260 265 270 Arg Glu MetThr Leu Ala Val Leu Asp Val Val Ala Leu Phe Pro Asn 275 280 285 Tyr AspThr Arg Thr Tyr Pro Met Glu Thr Lys Ala Gln Leu Thr Arg 290 295 300 GluVal Tyr Thr Asp Pro Leu Gly Ala Val Asn Val Ser Ser Ile Gly 305 310 315320 Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val Ile Glu Ser Ser Val 325330 335 Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr Gly Leu Thr Val Tyr340 345 350 Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr Ile Arg His TrpAla 355 360 365 Gly His Gln Ile Ser Tyr His Arg Val Ser Arg Gly Ser AsnLeu Gln 370 375 380 Gln Met Tyr Gly Thr Asn Gln Asn Leu His Ser Thr SerThr Phe Asp 385 390 395 400 Phe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu SerLys Asp Ala Val Leu 405 410 415 Leu Asp Ile Val Tyr Pro Gly Tyr Thr TyrIle Phe Phe Gly Met Pro 420 425 430 Glu Val Glu Phe Phe Met Val Asn GlnLeu Asn Asn Thr Arg Lys Thr 435 440 445 Leu Lys Tyr Asn Pro Val Ser LysAsp Ile Ile Ala Ser Thr Arg Asp 450 455 460 Ser Glu Leu Glu Leu Pro ProGlu Thr Ser Asp Gln Pro Asn Tyr Glu 465 470 475 480 Ser Tyr Ser His ArgLeu Cys His Ile Thr Ser Ile Pro Ala Thr Gly 485 490 495 Asn Thr Thr GlyLeu Val Pro Val Phe Ser Trp Thr His Arg Ser Ala 500 505 510 Asp Leu AsnAsn Thr Ile Tyr Ser Asp Lys Ile Thr Gln Ile Pro Ala 515 520 525 Val LysCys Trp Asp Asn Leu Pro Phe Val Pro Val Val Lys Gly Pro 530 535 540 GlyHis Thr Gly Gly Asp Leu Leu Gln Tyr Asn Arg Ser Thr Gly Ser 545 550 555560 Val Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu Ala Leu Glu Lys Ala 565570 575 Gly Lys Tyr Arg Val Arg Leu Arg Tyr Ala Thr Asp Ala Asp Ile Val580 585 590 Leu His Val Asn Asp Ala Gln Ile Gln Met Pro Lys Thr Met AsnPro 595 600 605 Gly Glu Asp Leu Thr Ser Lys Thr Phe Lys Val Ala Asp AlaIle Thr 610 615 620 Thr Leu Asn Leu Ala Thr Asp Ser Ser Leu Ala Leu LysHis Asn Leu 625 630 635 640 Gly Glu Asp Pro Asn Ser Thr Leu Ser Gly IleVal Tyr Val Asp Arg 645 650 655 Ile Glu Phe Ile Pro Val Asp Glu Thr TyrGlu 660 665 <210> SEQ ID NO 7 <211> LENGTH: 2003 <212> TYPE: DNA <213>ORGANISM: Bacillus thuringiensis (truncated) <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)...(2001) <221> NAME/KEY: misc_feature<222> LOCATION: (0)...(0) <223> OTHER INFORMATION: 1218-2 <400>SEQUENCE: 7 atg agt cca aat aat caa aat gaa tat gaa att ata gat gcg acacct 48 Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 15 10 15 tct act tct gta tcc aat gat tct aac aga tac cct ttt gcg aat gag96 Ser Thr Ser Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 2530 cca aca aat gcg cta caa aat atg gat tat aaa gat tat tta aaa atg 144Pro Thr Asn Ala Leu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45tct gcg gga aat gct agt gaa tac cct ggt tca cct gaa gta ctt gtt 192 SerAla Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 50 55 60 agcgga caa gat gca gct aag gcc gca att gat ata gta ggt aaa tta 240 Ser GlyGln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly Lys Leu 65 70 75 80 ctatca ggt tta ggg gtc cca ttt gtt ggg ccg ata gtg agt ctt tat 288 Leu SerGly Leu Gly Val Pro Phe Val Gly Pro Ile Val Ser Leu Tyr 85 90 95 act caactt att gat att ctg tgg cct tca ggg caa aag agt caa tgg 336 Thr Gln LeuIle Asp Ile Leu Trp Pro Ser Gly Gln Lys Ser Gln Trp 100 105 110 gag attttt atg gaa caa gta gaa gaa ctc ata aat caa aaa ata gca 384 Glu Ile PheMet Glu Gln Val Glu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 gaa tatgca agg aat aaa gcg ctt tcg gaa tta gaa gga tta ggt aat 432 Glu Tyr AlaArg Asn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 aat taccaa tta tat cta act gcg ctt gaa gaa tgg aaa gaa aat cca 480 Asn Tyr GlnLeu Tyr Leu Thr Ala Leu Glu Glu Trp Lys Glu Asn Pro 145 150 155 160 aatggt tca aga gcc tta cga gat gtg cga aat cga ttt gaa atc ctg 528 Asn GlySer Arg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu Ile Leu 165 170 175 gatagt tta ttt acg caa tac atg cca tct ttt cga gtg aca aat ttt 576 Asp SerLeu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn Phe 180 185 190 gaagta cca ttc ctt aca gta tat aca cag gca gcc aac ctt cat tta 624 Glu ValPro Phe Leu Thr Val Tyr Thr Gln Ala Ala Asn Leu His Leu 195 200 205 ctgtta tta aag gac gct tca att ttt gga gaa gaa tgg gga tgg tct 672 Leu LeuLeu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp Ser 210 215 220 acaacc act att aat aac tat tat gat cgt caa atg aaa ctt act gca 720 Thr ThrThr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu Thr Ala 225 230 235 240gaa tat tct gat cac tgt gta aag tgg tat gaa act ggt tta gca aaa 768 GluTyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu Ala Lys 245 250 255tta aaa ggc acg agc gct aaa caa tgg gtc gac tat aac caa ttc cgt 816 LeuLys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr Asn Gln Phe Arg 260 265 270aga gaa atg aca ctg acg gtt tta gat gtt gtt gca tta ttc cca aat 864 ArgGlu Met Thr Leu Thr Val Leu Asp Val Val Ala Leu Phe Pro Asn 275 280 285tat gac aca cgc acg tac cca atg gaa acg aaa gca caa cta aca agg 912 TyrAsp Thr Arg Thr Tyr Pro Met Glu Thr Lys Ala Gln Leu Thr Arg 290 295 300gaa gta tat aca gat cca ctg ggc gcg gta aac gtg tct tca att ggt 960 GluVal Tyr Thr Asp Pro Leu Gly Ala Val Asn Val Ser Ser Ile Gly 305 310 315320 tcc tgg tat gac aaa gca cct tct ttc gga gtg ata gaa tca tcc gtt 1008Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val Ile Glu Ser Ser Val 325 330335 att cga cca ccc cat gta ttt gat tat ata acg gga ctc aca gtg tat 1056Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr Gly Leu Thr Val Tyr 340 345350 aca caa tca aga agc att tct tcc gct cgc tat ata aga cat tgg gct 1104Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr Ile Arg His Trp Ala 355 360365 ggt cat caa ata agc tat cat cgg att ttt agt gat aat att ata aaa 1152Gly His Gln Ile Ser Tyr His Arg Ile Phe Ser Asp Asn Ile Ile Lys 370 375380 cag atg tat gga act aat caa aat cta cac agc act agt acc ttt gat 1200Gln Met Tyr Gly Thr Asn Gln Asn Leu His Ser Thr Ser Thr Phe Asp 385 390395 400 ttt acg aat tat gat att tac aag acg tta tca aaa gat gcg gtg ctc1248 Phe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405410 415 ctt gat att gtt ttt cct ggt tat acg tat ata ttt ttt gga atg cca1296 Leu Asp Ile Val Phe Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420425 430 gaa gtc gag ttt ttc atg gta aac caa ttg aat aat acc aga aag acg1344 Glu Val Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys Thr 435440 445 tta aag tat aat ccg gtt tcc aaa gat att ata gcg ggg aca aga gat1392 Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Gly Thr Arg Asp 450455 460 tcg gaa tta gaa tta cct cca gaa act tca gat caa cca aat tat gag1440 Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr Glu 465470 475 480 tca tat agc cat aga tta tgt cat atc aca agt att ccc gcg acgggt 1488 Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala Thr Gly485 490 495 tca act acc gga tta gta cct gta ttt tct tgg aca cat cgg agtgcc 1536 Ser Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr His Arg Ser Ala500 505 510 gat ctt ata aat gca gtt cat tca gat aaa att act cag att ccggtc 1584 Asp Leu Ile Asn Ala Val His Ser Asp Lys Ile Thr Gln Ile Pro Val515 520 525 gta aag gtt tct gat ttg gct ccc tct ata aca gga ggg cca aataat 1632 Val Lys Val Ser Asp Leu Ala Pro Ser Ile Thr Gly Gly Pro Asn Asn530 535 540 acc gtt gta tcg ggt cct gga ttt aca ggg ggg ggg ata ata aaagta 1680 Thr Val Val Ser Gly Pro Gly Phe Thr Gly Gly Gly Ile Ile Lys Val545 550 555 560 ata aga aat gga gta att ata tca cat atg cgt gtt aaa atttca gac 1728 Ile Arg Asn Gly Val Ile Ile Ser His Met Arg Val Lys Ile SerAsp 565 570 575 att aac aaa gaa tat agt atg agg att cgg tat gct tcc gctaat aat 1776 Ile Asn Lys Glu Tyr Ser Met Arg Ile Arg Tyr Ala Ser Ala AsnAsn 580 585 590 act gaa ttt tat ata aat cct tct gaa gaa aac gtt aaa tctcac gct 1824 Thr Glu Phe Tyr Ile Asn Pro Ser Glu Glu Asn Val Lys Ser HisAla 595 600 605 caa aaa act atg aat aga ggt gaa gct tta aca tat aat aaattt aat 1872 Gln Lys Thr Met Asn Arg Gly Glu Ala Leu Thr Tyr Asn Lys PheAsn 610 615 620 tat gcg act ttg ccc cct att aaa ttt acg aca acc gaa cctttc att 1920 Tyr Ala Thr Leu Pro Pro Ile Lys Phe Thr Thr Thr Glu Pro PheIle 625 630 635 640 act cta ggg gct ata ttt gaa gcg gaa gac ttt ctt ggaatt gaa gct 1968 Thr Leu Gly Ala Ile Phe Glu Ala Glu Asp Phe Leu Gly IleGlu Ala 645 650 655 tat ata gac cga atc gaa ttt atc cca gta gat ga 2003Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Asp 660 665 <210> SEQ ID NO 8<211> LENGTH: 667 <212> TYPE: PRT <213> ORGANISM: Bacillus thuringiensis(truncated) <400> SEQUENCE: 8 Met Ser Pro Asn Asn Gln Asn Glu Tyr GluIle Ile Asp Ala Thr Pro 1 5 10 15 Ser Thr Ser Val Ser Asn Asp Ser AsnArg Tyr Pro Phe Ala Asn Glu 20 25 30 Pro Thr Asn Ala Leu Gln Asn Met AspTyr Lys Asp Tyr Leu Lys Met 35 40 45 Ser Ala Gly Asn Ala Ser Glu Tyr ProGly Ser Pro Glu Val Leu Val 50 55 60 Ser Gly Gln Asp Ala Ala Lys Ala AlaIle Asp Ile Val Gly Lys Leu 65 70 75 80 Leu Ser Gly Leu Gly Val Pro PheVal Gly Pro Ile Val Ser Leu Tyr 85 90 95 Thr Gln Leu Ile Asp Ile Leu TrpPro Ser Gly Gln Lys Ser Gln Trp 100 105 110 Glu Ile Phe Met Glu Gln ValGlu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 Glu Tyr Ala Arg Asn LysAla Leu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 Asn Tyr Gln Leu TyrLeu Thr Ala Leu Glu Glu Trp Lys Glu Asn Pro 145 150 155 160 Asn Gly SerArg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu Ile Leu 165 170 175 Asp SerLeu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn Phe 180 185 190 GluVal Pro Phe Leu Thr Val Tyr Thr Gln Ala Ala Asn Leu His Leu 195 200 205Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp Ser 210 215220 Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu Thr Ala 225230 235 240 Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu AlaLys 245 250 255 Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr Asn GlnPhe Arg 260 265 270 Arg Glu Met Thr Leu Thr Val Leu Asp Val Val Ala LeuPhe Pro Asn 275 280 285 Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys AlaGln Leu Thr Arg 290 295 300 Glu Val Tyr Thr Asp Pro Leu Gly Ala Val AsnVal Ser Ser Ile Gly 305 310 315 320 Ser Trp Tyr Asp Lys Ala Pro Ser PheGly Val Ile Glu Ser Ser Val 325 330 335 Ile Arg Pro Pro His Val Phe AspTyr Ile Thr Gly Leu Thr Val Tyr 340 345 350 Thr Gln Ser Arg Ser Ile SerSer Ala Arg Tyr Ile Arg His Trp Ala 355 360 365 Gly His Gln Ile Ser TyrHis Arg Ile Phe Ser Asp Asn Ile Ile Lys 370 375 380 Gln Met Tyr Gly ThrAsn Gln Asn Leu His Ser Thr Ser Thr Phe Asp 385 390 395 400 Phe Thr AsnTyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405 410 415 Leu AspIle Val Phe Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420 425 430 GluVal Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys Thr 435 440 445Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Gly Thr Arg Asp 450 455460 Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr Glu 465470 475 480 Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala ThrGly 485 490 495 Ser Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr His ArgSer Ala 500 505 510 Asp Leu Ile Asn Ala Val His Ser Asp Lys Ile Thr GlnIle Pro Val 515 520 525 Val Lys Val Ser Asp Leu Ala Pro Ser Ile Thr GlyGly Pro Asn Asn 530 535 540 Thr Val Val Ser Gly Pro Gly Phe Thr Gly GlyGly Ile Ile Lys Val 545 550 555 560 Ile Arg Asn Gly Val Ile Ile Ser HisMet Arg Val Lys Ile Ser Asp 565 570 575 Ile Asn Lys Glu Tyr Ser Met ArgIle Arg Tyr Ala Ser Ala Asn Asn 580 585 590 Thr Glu Phe Tyr Ile Asn ProSer Glu Glu Asn Val Lys Ser His Ala 595 600 605 Gln Lys Thr Met Asn ArgGly Glu Ala Leu Thr Tyr Asn Lys Phe Asn 610 615 620 Tyr Ala Thr Leu ProPro Ile Lys Phe Thr Thr Thr Glu Pro Phe Ile 625 630 635 640 Thr Leu GlyAla Ile Phe Glu Ala Glu Asp Phe Leu Gly Ile Glu Ala 645 650 655 Tyr IleAsp Arg Ile Glu Phe Ile Pro Val Asp 660 665 <210> SEQ ID NO 9 <211>LENGTH: 2010 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(2010) <223> OTHERINFORMATION: Maize optimized Cry1218-1 <221> NAME/KEY: misc_feature<222> LOCATION: (0)...(0) <223> OTHER INFORMATION: mo1218-1 <400>SEQUENCE: 9 atg tcc ccc aac aac cag aac gag tac gag atc atc gac gcc accccc 48 Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 15 10 15 tcc acc tcc gtg tcc aac gac tcc aac cgc tac ccc ttc gcc aac gag96 Ser Thr Ser Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 2530 ccc acc aac gcc ctc cag aac atg gac tac aag gac tac ctc aag atg 144Pro Thr Asn Ala Leu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45tcc gcc ggc aac gcc tcc gag tac ccc ggc tcc ccc gag gtg ctc gtg 192 SerAla Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 50 55 60 tccggc cag gac gcc gcc aag gcc gcc atc gac atc gtg ggc aag ctc 240 Ser GlyGln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly Lys Leu 65 70 75 80 ctctcc ggc ctc ggc gtg ccc ttc gtg ggc ccc atc gtg tcc ctc tac 288 Leu SerGly Leu Gly Val Pro Phe Val Gly Pro Ile Val Ser Leu Tyr 85 90 95 acc cagctc atc gac atc ctc tgg ccc tcc ggc gag aag tcc cag tgg 336 Thr Gln LeuIle Asp Ile Leu Trp Pro Ser Gly Glu Lys Ser Gln Trp 100 105 110 gaa atcttc atg gag cag gtg gag gag ctc atc aac cag aag atc gcc 384 Glu Ile PheMet Glu Gln Val Glu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 gag tacgcc cgc aac aag gcc ctc tcc gag ctg gag ggc ctc ggc aac 432 Glu Tyr AlaArg Asn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 aac taccag ctc tac ctc acc gcc ctg gag gag tgg gag gag aac ccc 480 Asn Tyr GlnLeu Tyr Leu Thr Ala Leu Glu Glu Trp Glu Glu Asn Pro 145 150 155 160 aacggc tcc cgc gcc ctc cgc gac gtg cgc aac cgc ttc gag atc ctc 528 Asn GlySer Arg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu Ile Leu 165 170 175 gactcc ctc ttc acc cag tac atg ccc tcc ttc cgc gtg acc aac ttc 576 Asp SerLeu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn Phe 180 185 190 gaggtg ccc ttc ctc acc gtg tac gcc atg gcc gcc aac ctc cac ctc 624 Glu ValPro Phe Leu Thr Val Tyr Ala Met Ala Ala Asn Leu His Leu 195 200 205 ctcctc ctc aag gac gcc tcc atc ttc ggc gag gag tgg ggc tgg tcc 672 Leu LeuLeu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp Ser 210 215 220 accacc acc atc aac aac tac tac gac cgc cag atg aag ctc acc gcc 720 Thr ThrThr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu Thr Ala 225 230 235 240gag tac tcc gac cac tgc gtg aag tgg tat gag acc ggc ctc gcc aag 768 GluTyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu Ala Lys 245 250 255ctc aag ggc acc tcc gcc aag cag tgg gtg gac tac aac cag ttc cgc 816 LeuLys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr Asn Gln Phe Arg 260 265 270cgc gag atg acc ctc gcc gtg ctc gac gtg gtg gcc ctc ttc ccc aac 864 ArgGlu Met Thr Leu Ala Val Leu Asp Val Val Ala Leu Phe Pro Asn 275 280 285tac gac acc cgc acc tac ccc atg gag acc aag gcc cag ctc acc cgc 912 TyrAsp Thr Arg Thr Tyr Pro Met Glu Thr Lys Ala Gln Leu Thr Arg 290 295 300gag gtg tac acc gac ccg ctc ggc gcc gtg aac gtg tcc tcc atc ggc 960 GluVal Tyr Thr Asp Pro Leu Gly Ala Val Asn Val Ser Ser Ile Gly 305 310 315320 tct tgg tac gac aag gcc cca agc ttc ggc gtg atc gag tcc tcc gtg 1008Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val Ile Glu Ser Ser Val 325 330335 atc cgc ccg ccg cac gtg ttc gac tac atc acc ggc ctc acc gtg tac 1056Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr Gly Leu Thr Val Tyr 340 345350 acc cag tcc cgc tcc atc tcc tcc gcc cgc tac atc cgc cac tgg gcc 1104Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr Ile Arg His Trp Ala 355 360365 ggc cac cag atc tcc tac cac cgc gtg tcc cgc ggc tcc aac ctc cag 1152Gly His Gln Ile Ser Tyr His Arg Val Ser Arg Gly Ser Asn Leu Gln 370 375380 cag atg tac ggc acc aac cag aac ctc cac tcc acc tcc acc ttc gac 1200Gln Met Tyr Gly Thr Asn Gln Asn Leu His Ser Thr Ser Thr Phe Asp 385 390395 400 ttc acc aac tac gac atc tac aag acc ctc tcc aag gac gcc gtg ctc1248 Phe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405410 415 ctc gac atc gtg tac ccc ggc tac acc tac atc ttc ttc ggc atg ccg1296 Leu Asp Ile Val Tyr Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420425 430 gag gtg gag ttc ttc atg gtg aac cag ctc aac aac acc cgc aag acc1344 Glu Val Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys Thr 435440 445 ctc aaa tac aac ccc gtg tcc aag gac atc atc gcc tcc acc cgc gac1392 Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Ser Thr Arg Asp 450455 460 tcc gag ctc gag ctc ccc ccc gag acc tcc gac cag ccc aac tac gag1440 Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr Glu 465470 475 480 tcc tac tcc cac cgc ctc tgc cac atc acc tcc atc ccc gcc accggc 1488 Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala Thr Gly485 490 495 aac acc acc ggc ctc gtg ccg gtg ttc tcc tgg acc cac cgc tctgca 1536 Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr His Arg Ser Ala500 505 510 gac ctc aac aac acc atc tac tcc gac aag atc acc cag atc cccgcc 1584 Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile Thr Gln Ile Pro Ala515 520 525 gtg aag tgc tgg gac aac ctc ccc ttc gtg ccc gtg gtg aag ggcccc 1632 Val Lys Cys Trp Asp Asn Leu Pro Phe Val Pro Val Val Lys Gly Pro530 535 540 ggc cac acc ggc ggc gac ctc ctc cag tac aac cgc tcc acc ggctcc 1680 Gly His Thr Gly Gly Asp Leu Leu Gln Tyr Asn Arg Ser Thr Gly Ser545 550 555 560 gtg ggc acc ctc ttc ctc gcc cgc tac ggc ctc gcc ctg gagaag gcc 1728 Val Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu Ala Leu Glu LysAla 565 570 575 ggc aag tac cgc gtg cgc ctc cgc tac gcc act gac gcc gacatc gtg 1776 Gly Lys Tyr Arg Val Arg Leu Arg Tyr Ala Thr Asp Ala Asp IleVal 580 585 590 ctc cac gtg aac gac gcc cag atc cag atg ccc aag acc atgaac ccc 1824 Leu His Val Asn Asp Ala Gln Ile Gln Met Pro Lys Thr Met AsnPro 595 600 605 ggc gag gac ctc acc tcc aag acc ttc aag gtg gcc gac gccatc acc 1872 Gly Glu Asp Leu Thr Ser Lys Thr Phe Lys Val Ala Asp Ala IleThr 610 615 620 acc ctc aac ctc gcc acc gac tcc tcc ctc gcc ctc aag cacaac ctc 1920 Thr Leu Asn Leu Ala Thr Asp Ser Ser Leu Ala Leu Lys His AsnLeu 625 630 635 640 ggc gag gac ccc aac tcc acc ctc tcc ggc atc gtg tacgtg gac cgc 1968 Gly Glu Asp Pro Asn Ser Thr Leu Ser Gly Ile Val Tyr ValAsp Arg 645 650 655 atc gag ttc atc ccc gtg gac gag acc tac gag gcc gagtga 2010 Ile Glu Phe Ile Pro Val Asp Glu Thr Tyr Glu Ala Glu * 660 665<210> SEQ ID NO 10 <211> LENGTH: 669 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Maizeoptimized Cry1218-1 <400> SEQUENCE: 10 Met Ser Pro Asn Asn Gln Asn GluTyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 Ser Thr Ser Val Ser Asn AspSer Asn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 Pro Thr Asn Ala Leu Gln AsnMet Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45 Ser Ala Gly Asn Ala Ser GluTyr Pro Gly Ser Pro Glu Val Leu Val 50 55 60 Ser Gly Gln Asp Ala Ala LysAla Ala Ile Asp Ile Val Gly Lys Leu 65 70 75 80 Leu Ser Gly Leu Gly ValPro Phe Val Gly Pro Ile Val Ser Leu Tyr 85 90 95 Thr Gln Leu Ile Asp IleLeu Trp Pro Ser Gly Glu Lys Ser Gln Trp 100 105 110 Glu Ile Phe Met GluGln Val Glu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 Glu Tyr Ala ArgAsn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 Asn Tyr GlnLeu Tyr Leu Thr Ala Leu Glu Glu Trp Glu Glu Asn Pro 145 150 155 160 AsnGly Ser Arg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu Ile Leu 165 170 175Asp Ser Leu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn Phe 180 185190 Glu Val Pro Phe Leu Thr Val Tyr Ala Met Ala Ala Asn Leu His Leu 195200 205 Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp Ser210 215 220 Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu ThrAla 225 230 235 240 Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr GlyLeu Ala Lys 245 250 255 Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp TyrAsn Gln Phe Arg 260 265 270 Arg Glu Met Thr Leu Ala Val Leu Asp Val ValAla Leu Phe Pro Asn 275 280 285 Tyr Asp Thr Arg Thr Tyr Pro Met Glu ThrLys Ala Gln Leu Thr Arg 290 295 300 Glu Val Tyr Thr Asp Pro Leu Gly AlaVal Asn Val Ser Ser Ile Gly 305 310 315 320 Ser Trp Tyr Asp Lys Ala ProSer Phe Gly Val Ile Glu Ser Ser Val 325 330 335 Ile Arg Pro Pro His ValPhe Asp Tyr Ile Thr Gly Leu Thr Val Tyr 340 345 350 Thr Gln Ser Arg SerIle Ser Ser Ala Arg Tyr Ile Arg His Trp Ala 355 360 365 Gly His Gln IleSer Tyr His Arg Val Ser Arg Gly Ser Asn Leu Gln 370 375 380 Gln Met TyrGly Thr Asn Gln Asn Leu His Ser Thr Ser Thr Phe Asp 385 390 395 400 PheThr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405 410 415Leu Asp Ile Val Tyr Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420 425430 Glu Val Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys Thr 435440 445 Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Ser Thr Arg Asp450 455 460 Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn TyrGlu 465 470 475 480 Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile ProAla Thr Gly 485 490 495 Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp ThrHis Arg Ser Ala 500 505 510 Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys IleThr Gln Ile Pro Ala 515 520 525 Val Lys Cys Trp Asp Asn Leu Pro Phe ValPro Val Val Lys Gly Pro 530 535 540 Gly His Thr Gly Gly Asp Leu Leu GlnTyr Asn Arg Ser Thr Gly Ser 545 550 555 560 Val Gly Thr Leu Phe Leu AlaArg Tyr Gly Leu Ala Leu Glu Lys Ala 565 570 575 Gly Lys Tyr Arg Val ArgLeu Arg Tyr Ala Thr Asp Ala Asp Ile Val 580 585 590 Leu His Val Asn AspAla Gln Ile Gln Met Pro Lys Thr Met Asn Pro 595 600 605 Gly Glu Asp LeuThr Ser Lys Thr Phe Lys Val Ala Asp Ala Ile Thr 610 615 620 Thr Leu AsnLeu Ala Thr Asp Ser Ser Leu Ala Leu Lys His Asn Leu 625 630 635 640 GlyGlu Asp Pro Asn Ser Thr Leu Ser Gly Ile Val Tyr Val Asp Arg 645 650 655Ile Glu Phe Ile Pro Val Asp Glu Thr Tyr Glu Ala Glu 660 665 <210> SEQ IDNO 11 <211> LENGTH: 2022 <212> TYPE: DNA <213> ORGANISM: Bacillusthuringiensis (mutated) <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)...(2022) <221> NAME/KEY: misc_feature <222> LOCATION:(0)...(0) <223> OTHER INFORMATION: NGSR.N1218-1 <400> SEQUENCE: 11 atgagt cca aat aat caa aat gaa tat gaa att ata gat gcg aca cct 48 Met SerPro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 tctact tct gta tcc aat gat tct aac aga tac cct ttt gcg aat gag 96 Ser ThrSer Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 cca acaaat gcg cta caa aat atg gat tat aaa gat tat tta aaa atg 144 Pro Thr AsnAla Leu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45 tct gcg ggaaat gct agt gaa tac cct ggt tca cct gaa gta ctt gtt 192 Ser Ala Gly AsnAla Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 50 55 60 agc gga caa gatgca gct aag gcc gca att gat ata gta ggt aaa tta 240 Ser Gly Gln Asp AlaAla Lys Ala Ala Ile Asp Ile Val Gly Lys Leu 65 70 75 80 cta tca ggt ttaggg gtc cca ttt gtt ggg ccg ata gtg agt ctt tat 288 Leu Ser Gly Leu GlyVal Pro Phe Val Gly Pro Ile Val Ser Leu Tyr 85 90 95 act caa ctt att gatatt ctg tgg cct tca ggg gaa aag agt caa tgg 336 Thr Gln Leu Ile Asp IleLeu Trp Pro Ser Gly Glu Lys Ser Gln Trp 100 105 110 gaa att ttt atg gaacaa gta gaa gaa ctc att aat caa aaa ata gca 384 Glu Ile Phe Met Glu GlnVal Glu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 gaa tat gca agg aataaa gcg ctt tcg gaa tta gaa gga tta ggt aat 432 Glu Tyr Ala Arg Asn LysAla Leu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 aat tac caa tta tatcta act gcg ctt gaa gaa tgg gaa gaa aat cca 480 Asn Tyr Gln Leu Tyr LeuThr Ala Leu Glu Glu Trp Glu Glu Asn Pro 145 150 155 160 aat ggt tca agaaat ggt tcc cgg gcc tta cga gat gtg cga aat cga 528 Asn Gly Ser Arg AsnGly Ser Arg Ala Leu Arg Asp Val Arg Asn Arg 165 170 175 ttt gaa atc ctggat agt tta ttt acg caa tat atg cca tct ttt aga 576 Phe Glu Ile Leu AspSer Leu Phe Thr Gln Tyr Met Pro Ser Phe Arg 180 185 190 gtg aca aat tttgaa gta cca ttc ctt act gta tat gca atg gca gcc 624 Val Thr Asn Phe GluVal Pro Phe Leu Thr Val Tyr Ala Met Ala Ala 195 200 205 aac ctt cat ttactg tta tta aag gac gcg tca att ttt gga gaa gaa 672 Asn Leu His Leu LeuLeu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu 210 215 220 tgg gga tgg tcaaca act act att aat aac tat tat gat cgt caa atg 720 Trp Gly Trp Ser ThrThr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met 225 230 235 240 aaa ctt actgca gaa tat tct gat cac tgt gta aag tgg tat gaa act 768 Lys Leu Thr AlaGlu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr 245 250 255 ggt tta gcaaaa tta aaa ggc acg agc gct aaa caa tgg gtt gac tat 816 Gly Leu Ala LysLeu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr 260 265 270 aac caa ttccgt aga gaa atg aca ctg gcg gtt tta gat gtt gtt gca 864 Asn Gln Phe ArgArg Glu Met Thr Leu Ala Val Leu Asp Val Val Ala 275 280 285 tta ttc ccaaat tat gac aca cgc acg tac cca atg gaa acg aaa gca 912 Leu Phe Pro AsnTyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys Ala 290 295 300 caa cta acaagg gaa gta tat aca gat cca ctg ggc gcg gta aac gtg 960 Gln Leu Thr ArgGlu Val Tyr Thr Asp Pro Leu Gly Ala Val Asn Val 305 310 315 320 tct tcaatt ggt tcc tgg tat gac aaa gca cct tct ttc gga gtg ata 1008 Ser Ser IleGly Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val Ile 325 330 335 gaa tcatcc gtt att cga cca ccc cat gta ttt gat tat ata acg gga 1056 Glu Ser SerVal Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr Gly 340 345 350 ctc acagtg tat aca caa tca aga agc att tct tcc gct cgc tat ata 1104 Leu Thr ValTyr Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr Ile 355 360 365 aga cattgg gct ggt cat caa ata agc tac cat cgt gtc agt agg ggt 1152 Arg His TrpAla Gly His Gln Ile Ser Tyr His Arg Val Ser Arg Gly 370 375 380 agt aatctt caa caa atg tat gga act aat caa aat cta cac agc act 1200 Ser Asn LeuGln Gln Met Tyr Gly Thr Asn Gln Asn Leu His Ser Thr 385 390 395 400 agtacc ttt gat ttt acg aat tat gat att tac aag act cta tca aag 1248 Ser ThrPhe Asp Phe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser Lys 405 410 415 gatgca gta ctc ctt gat att gtt tac cct ggt tat acg tat ata ttt 1296 Asp AlaVal Leu Leu Asp Ile Val Tyr Pro Gly Tyr Thr Tyr Ile Phe 420 425 430 tttgga atg cca gaa gtc gag ttt ttc atg gta aac caa ttg aat aat 1344 Phe GlyMet Pro Glu Val Glu Phe Phe Met Val Asn Gln Leu Asn Asn 435 440 445 accaga aag acg tta aag tat aat cca gtt tcc aaa gat att ata gcg 1392 Thr ArgLys Thr Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala 450 455 460 agtaca aga gat tcg gaa tta gaa tta cct cca gaa act tca gat caa 1440 Ser ThrArg Asp Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln 465 470 475 480cca aat tat gag tca tat agc cat aga tta tgt cat atc aca agt att 1488 ProAsn Tyr Glu Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile 485 490 495ccc gcg acg ggt aac act acc gga tta gta cct gta ttt tct tgg aca 1536 ProAla Thr Gly Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr 500 505 510cat cga agt gca gat tta aac aat aca ata tat tca gat aaa atc act 1584 HisArg Ser Ala Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile Thr 515 520 525caa att ccg gcc gtt aaa tgt tgg gat aat tta ccg ttt gtt cca gtg 1632 GlnIle Pro Ala Val Lys Cys Trp Asp Asn Leu Pro Phe Val Pro Val 530 535 540gta aaa gga cca gga cat aca gga ggg gat tta tta cag tat aat aga 1680 ValLys Gly Pro Gly His Thr Gly Gly Asp Leu Leu Gln Tyr Asn Arg 545 550 555560 agt act ggt tct gta gga acc tta ttt cta gct cga tat ggc cta gca 1728Ser Thr Gly Ser Val Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu Ala 565 570575 tta gaa aaa gca ggg aaa tat cgt gta aga ctg aga tat gct act gat 1776Leu Glu Lys Ala Gly Lys Tyr Arg Val Arg Leu Arg Tyr Ala Thr Asp 580 585590 gca gat att gta ttg cat gta aac gat gct cag att cag atg cca aaa 1824Ala Asp Ile Val Leu His Val Asn Asp Ala Gln Ile Gln Met Pro Lys 595 600605 aca atg aac cca ggt gag gat ctg aca tct aaa act ttt aaa gtt gca 1872Thr Met Asn Pro Gly Glu Asp Leu Thr Ser Lys Thr Phe Lys Val Ala 610 615620 gat gct atc aca aca tta aat tta gca aca gat agt tcg cta gca ttg 1920Asp Ala Ile Thr Thr Leu Asn Leu Ala Thr Asp Ser Ser Leu Ala Leu 625 630635 640 aaa cat aat tta ggt gaa gac cct aat tca aca tta tct ggt ata gtt1968 Lys His Asn Leu Gly Glu Asp Pro Asn Ser Thr Leu Ser Gly Ile Val 645650 655 tac gtt gac cga atc gaa ttc atc cca gta gat gag aca tat gaa gcg2016 Tyr Val Asp Arg Ile Glu Phe Ile Pro Val Asp Glu Thr Tyr Glu Ala 660665 670 gaa taa 2022 Glu * <210> SEQ ID NO 12 <211> LENGTH: 673 <212>TYPE: PRT <213> ORGANISM: Bacillus thuringiensis (mutated) <400>SEQUENCE: 12 Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala ThrPro 1 5 10 15 Ser Thr Ser Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe AlaAsn Glu 20 25 30 Pro Thr Asn Ala Leu Gln Asn Met Asp Tyr Lys Asp Tyr LeuLys Met 35 40 45 Ser Ala Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu ValLeu Val 50 55 60 Ser Gly Gln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val GlyLys Leu 65 70 75 80 Leu Ser Gly Leu Gly Val Pro Phe Val Gly Pro Ile ValSer Leu Tyr 85 90 95 Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly Glu LysSer Gln Trp 100 105 110 Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile AsnGln Lys Ile Ala 115 120 125 Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu LeuGlu Gly Leu Gly Asn 130 135 140 Asn Tyr Gln Leu Tyr Leu Thr Ala Leu GluGlu Trp Glu Glu Asn Pro 145 150 155 160 Asn Gly Ser Arg Asn Gly Ser ArgAla Leu Arg Asp Val Arg Asn Arg 165 170 175 Phe Glu Ile Leu Asp Ser LeuPhe Thr Gln Tyr Met Pro Ser Phe Arg 180 185 190 Val Thr Asn Phe Glu ValPro Phe Leu Thr Val Tyr Ala Met Ala Ala 195 200 205 Asn Leu His Leu LeuLeu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu 210 215 220 Trp Gly Trp SerThr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met 225 230 235 240 Lys LeuThr Ala Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr 245 250 255 GlyLeu Ala Lys Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr 260 265 270Asn Gln Phe Arg Arg Glu Met Thr Leu Ala Val Leu Asp Val Val Ala 275 280285 Leu Phe Pro Asn Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys Ala 290295 300 Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Gly Ala Val Asn Val305 310 315 320 Ser Ser Ile Gly Ser Trp Tyr Asp Lys Ala Pro Ser Phe GlyVal Ile 325 330 335 Glu Ser Ser Val Ile Arg Pro Pro His Val Phe Asp TyrIle Thr Gly 340 345 350 Leu Thr Val Tyr Thr Gln Ser Arg Ser Ile Ser SerAla Arg Tyr Ile 355 360 365 Arg His Trp Ala Gly His Gln Ile Ser Tyr HisArg Val Ser Arg Gly 370 375 380 Ser Asn Leu Gln Gln Met Tyr Gly Thr AsnGln Asn Leu His Ser Thr 385 390 395 400 Ser Thr Phe Asp Phe Thr Asn TyrAsp Ile Tyr Lys Thr Leu Ser Lys 405 410 415 Asp Ala Val Leu Leu Asp IleVal Tyr Pro Gly Tyr Thr Tyr Ile Phe 420 425 430 Phe Gly Met Pro Glu ValGlu Phe Phe Met Val Asn Gln Leu Asn Asn 435 440 445 Thr Arg Lys Thr LeuLys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala 450 455 460 Ser Thr Arg AspSer Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln 465 470 475 480 Pro AsnTyr Glu Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile 485 490 495 ProAla Thr Gly Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr 500 505 510His Arg Ser Ala Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile Thr 515 520525 Gln Ile Pro Ala Val Lys Cys Trp Asp Asn Leu Pro Phe Val Pro Val 530535 540 Val Lys Gly Pro Gly His Thr Gly Gly Asp Leu Leu Gln Tyr Asn Arg545 550 555 560 Ser Thr Gly Ser Val Gly Thr Leu Phe Leu Ala Arg Tyr GlyLeu Ala 565 570 575 Leu Glu Lys Ala Gly Lys Tyr Arg Val Arg Leu Arg TyrAla Thr Asp 580 585 590 Ala Asp Ile Val Leu His Val Asn Asp Ala Gln IleGln Met Pro Lys 595 600 605 Thr Met Asn Pro Gly Glu Asp Leu Thr Ser LysThr Phe Lys Val Ala 610 615 620 Asp Ala Ile Thr Thr Leu Asn Leu Ala ThrAsp Ser Ser Leu Ala Leu 625 630 635 640 Lys His Asn Leu Gly Glu Asp ProAsn Ser Thr Leu Ser Gly Ile Val 645 650 655 Tyr Val Asp Arg Ile Glu PheIle Pro Val Asp Glu Thr Tyr Glu Ala 660 665 670 Glu <210> SEQ ID NO 13<211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: NGSR Insert <400> SEQUENCE: 13aatggttccc gg 12 <210> SEQ ID NO 14 <211> LENGTH: 4 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: NGSR Insert <400> SEQUENCE: 14 Asn Gly Ser Arg 1 <210> SEQID NO 15 <211> LENGTH: 2010 <212> TYPE: DNA <213> ORGANISM: Bacillusthuringiensis (truncated) <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)...(2010) <221> NAME/KEY: misc_feature <222> LOCATION:(0)...(0) <223> OTHER INFORMATION: 1218-1A <400> SEQUENCE: 15 atg agtcca aat aat caa aat gaa tat gaa att ata gat gcg aca cct 48 Met Ser ProAsn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 tct acttct gta tcc aat gat tct aac aga tac cct ttt gcg aat gag 96 Ser Thr SerVal Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 cca aca aatgcg cta caa aat atg gat tat aaa gat tat tta aaa atg 144 Pro Thr Asn AlaLeu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45 tct gcg gga aatgct agt gaa tac cct ggt tca cct gaa gta ctt gtt 192 Ser Ala Gly Asn AlaSer Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 50 55 60 agc gga caa gat gcagct aag gcc gca att gat ata gta ggt aaa tta 240 Ser Gly Gln Asp Ala AlaLys Ala Ala Ile Asp Ile Val Gly Lys Leu 65 70 75 80 cta tca ggt tta ggggtc cca ttt gtt ggg ccg ata gtg agt ctt tat 288 Leu Ser Gly Leu Gly ValPro Phe Val Gly Pro Ile Val Ser Leu Tyr 85 90 95 act caa ctt att gat attctg tgg cct tca ggg gaa aag agt caa tgg 336 Thr Gln Leu Ile Asp Ile LeuTrp Pro Ser Gly Glu Lys Ser Gln Trp 100 105 110 gaa att ttt atg gaa caagta gaa gaa ctc att aat caa aaa ata gca 384 Glu Ile Phe Met Glu Gln ValGlu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 gaa tat gca agg aat aaagcg ctt tcg gaa tta gaa gga tta ggt aat 432 Glu Tyr Ala Arg Asn Lys AlaLeu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 aat tac caa tta tat ctaact gcg ctt gaa gaa tgg gaa gaa aat cca 480 Asn Tyr Gln Leu Tyr Leu ThrAla Leu Glu Glu Trp Glu Glu Asn Pro 145 150 155 160 aat ggt tca aga gcctta cga gat gtg cga aat cga ttt gaa atc ctg 528 Asn Gly Ser Arg Ala LeuArg Asp Val Arg Asn Arg Phe Glu Ile Leu 165 170 175 gat agt tta ttt acgcaa tat atg cca tct ttt aga gtg aca aat ttt 576 Asp Ser Leu Phe Thr GlnTyr Met Pro Ser Phe Arg Val Thr Asn Phe 180 185 190 gaa gta cca ttc cttact gta tat gca atg gca gcc aac ctt cat tta 624 Glu Val Pro Phe Leu ThrVal Tyr Ala Met Ala Ala Asn Leu His Leu 195 200 205 ctg tta tta aag gacgcg tca att ttt gga gaa gaa tgg gga tgg tca 672 Leu Leu Leu Lys Asp AlaSer Ile Phe Gly Glu Glu Trp Gly Trp Ser 210 215 220 aca act act att aataac tat tat gat cgt caa atg aaa ctt act gca 720 Thr Thr Thr Ile Asn AsnTyr Tyr Asp Arg Gln Met Lys Leu Thr Ala 225 230 235 240 gaa tat tct gatcac tgt gta aag tgg tat gaa act ggt tta gca aaa 768 Glu Tyr Ser Asp HisCys Val Lys Trp Tyr Glu Thr Gly Leu Ala Lys 245 250 255 tta aaa ggc acgagc gct aaa caa tgg gtt gac tat aac caa ttc cgt 816 Leu Lys Gly Thr SerAla Lys Gln Trp Val Asp Tyr Asn Gln Phe Arg 260 265 270 aga gaa atg acactg gcg gtt tta gat gtt gtt gca tta ttc cca aat 864 Arg Glu Met Thr LeuAla Val Leu Asp Val Val Ala Leu Phe Pro Asn 275 280 285 tat gac aca cgcacg tac cca atg gaa acg aaa gca caa cta aca agg 912 Tyr Asp Thr Arg ThrTyr Pro Met Glu Thr Lys Ala Gln Leu Thr Arg 290 295 300 gaa gta tat acagat cca ctg ggc gcg gta aac gtg tct tca att ggt 960 Glu Val Tyr Thr AspPro Leu Gly Ala Val Asn Val Ser Ser Ile Gly 305 310 315 320 tcc tgg tatgac aaa gca cct tct ttc gga gtg ata gaa tca tcc gtt 1008 Ser Trp Tyr AspLys Ala Pro Ser Phe Gly Val Ile Glu Ser Ser Val 325 330 335 att cga ccaccc cat gta ttt gat tat ata acg gga ctc aca gtg tat 1056 Ile Arg Pro ProHis Val Phe Asp Tyr Ile Thr Gly Leu Thr Val Tyr 340 345 350 aca caa tcaaga agc att tct tcc gct cgc tat ata aga cat tgg gct 1104 Thr Gln Ser ArgSer Ile Ser Ser Ala Arg Tyr Ile Arg His Trp Ala 355 360 365 ggt cat caaata agc tac cat cgt gtc agt agg ggt agt aat ctt caa 1152 Gly His Gln IleSer Tyr His Arg Val Ser Arg Gly Ser Asn Leu Gln 370 375 380 caa atg tatgga act aat caa aat cta cac agc act agt acc ttt gat 1200 Gln Met Tyr GlyThr Asn Gln Asn Leu His Ser Thr Ser Thr Phe Asp 385 390 395 400 ttt acgaat tat gat att tac aag act cta tca aag gat gca gta ctc 1248 Phe Thr AsnTyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405 410 415 ctt gatatt gtt tac cct ggt tat acg tat ata ttt ttt gga atg cca 1296 Leu Asp IleVal Tyr Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420 425 430 gaa gtcgag ttt ttc atg gta aac caa ttg aat aat acc aga aag acg 1344 Glu Val GluPhe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys Thr 435 440 445 tta aagtat aat cca gtt tcc aaa gat att ata gcg agt aca aga gat 1392 Leu Lys TyrAsn Pro Val Ser Lys Asp Ile Ile Ala Ser Thr Arg Asp 450 455 460 tcg gaatta gaa tta cct cca gaa act tca gat caa cca aat tat gag 1440 Ser Glu LeuGlu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr Glu 465 470 475 480 tcatat agc cat aga tta tgt cat atc aca agt att ccc gcg acg ggt 1488 Ser TyrSer His Arg Leu Cys His Ile Thr Ser Ile Pro Ala Thr Gly 485 490 495 aacact acc gga tta gta cct gta ttt tct tgg aca cat cga agt gca 1536 Asn ThrThr Gly Leu Val Pro Val Phe Ser Trp Thr His Arg Ser Ala 500 505 510 gattta aac aat aca ata tat tca gat aaa atc act caa att ccg gcc 1584 Asp LeuAsn Asn Thr Ile Tyr Ser Asp Lys Ile Thr Gln Ile Pro Ala 515 520 525 gttaaa tgt tgg gat aat tta ccg ttt gtt cca gtg gta aaa gga cca 1632 Val LysCys Trp Asp Asn Leu Pro Phe Val Pro Val Val Lys Gly Pro 530 535 540 ggacat aca gga ggg gat tta tta cag tat aat aga agt act ggt tct 1680 Gly HisThr Gly Gly Asp Leu Leu Gln Tyr Asn Arg Ser Thr Gly Ser 545 550 555 560gta gga acc tta ttt cta gct cga tat ggc cta gca tta gaa aaa gca 1728 ValGly Thr Leu Phe Leu Ala Arg Tyr Gly Leu Ala Leu Glu Lys Ala 565 570 575ggg aaa tat cgt gta aga ctg aga tat gct act gat gca gat att gta 1776 GlyLys Tyr Arg Val Arg Leu Arg Tyr Ala Thr Asp Ala Asp Ile Val 580 585 590ttg cat gta aac gat gct cag att cag atg cca aaa aca atg aac cca 1824 LeuHis Val Asn Asp Ala Gln Ile Gln Met Pro Lys Thr Met Asn Pro 595 600 605ggt gag gat ctg aca tct aaa act ttt aaa gtt gca gat gct atc aca 1872 GlyGlu Asp Leu Thr Ser Lys Thr Phe Lys Val Ala Asp Ala Ile Thr 610 615 620aca tta aat tta gca aca gat agt tcg cta gca ttg aaa cat aat tta 1920 ThrLeu Asn Leu Ala Thr Asp Ser Ser Leu Ala Leu Lys His Asn Leu 625 630 635640 ggt gaa gac cct aat tca aca tta tct ggt ata gtt tac gtt gac cga 1968Gly Glu Asp Pro Asn Ser Thr Leu Ser Gly Ile Val Tyr Val Asp Arg 645 650655 atc gaa ttc atc cca gta gat gag aca tat gaa gcg gaa taa 2010 Ile GluPhe Ile Pro Val Asp Glu Thr Tyr Glu Ala Glu * 660 665 <210> SEQ ID NO 16<211> LENGTH: 669 <212> TYPE: PRT <213> ORGANISM: Bacillus thuringiensis(truncated) <400> SEQUENCE: 16 Met Ser Pro Asn Asn Gln Asn Glu Tyr GluIle Ile Asp Ala Thr Pro 1 5 10 15 Ser Thr Ser Val Ser Asn Asp Ser AsnArg Tyr Pro Phe Ala Asn Glu 20 25 30 Pro Thr Asn Ala Leu Gln Asn Met AspTyr Lys Asp Tyr Leu Lys Met 35 40 45 Ser Ala Gly Asn Ala Ser Glu Tyr ProGly Ser Pro Glu Val Leu Val 50 55 60 Ser Gly Gln Asp Ala Ala Lys Ala AlaIle Asp Ile Val Gly Lys Leu 65 70 75 80 Leu Ser Gly Leu Gly Val Pro PheVal Gly Pro Ile Val Ser Leu Tyr 85 90 95 Thr Gln Leu Ile Asp Ile Leu TrpPro Ser Gly Glu Lys Ser Gln Trp 100 105 110 Glu Ile Phe Met Glu Gln ValGlu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 Glu Tyr Ala Arg Asn LysAla Leu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 Asn Tyr Gln Leu TyrLeu Thr Ala Leu Glu Glu Trp Glu Glu Asn Pro 145 150 155 160 Asn Gly SerArg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu Ile Leu 165 170 175 Asp SerLeu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn Phe 180 185 190 GluVal Pro Phe Leu Thr Val Tyr Ala Met Ala Ala Asn Leu His Leu 195 200 205Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp Ser 210 215220 Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu Thr Ala 225230 235 240 Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu AlaLys 245 250 255 Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr Asn GlnPhe Arg 260 265 270 Arg Glu Met Thr Leu Ala Val Leu Asp Val Val Ala LeuPhe Pro Asn 275 280 285 Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys AlaGln Leu Thr Arg 290 295 300 Glu Val Tyr Thr Asp Pro Leu Gly Ala Val AsnVal Ser Ser Ile Gly 305 310 315 320 Ser Trp Tyr Asp Lys Ala Pro Ser PheGly Val Ile Glu Ser Ser Val 325 330 335 Ile Arg Pro Pro His Val Phe AspTyr Ile Thr Gly Leu Thr Val Tyr 340 345 350 Thr Gln Ser Arg Ser Ile SerSer Ala Arg Tyr Ile Arg His Trp Ala 355 360 365 Gly His Gln Ile Ser TyrHis Arg Val Ser Arg Gly Ser Asn Leu Gln 370 375 380 Gln Met Tyr Gly ThrAsn Gln Asn Leu His Ser Thr Ser Thr Phe Asp 385 390 395 400 Phe Thr AsnTyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405 410 415 Leu AspIle Val Tyr Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420 425 430 GluVal Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys Thr 435 440 445Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Ser Thr Arg Asp 450 455460 Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr Glu 465470 475 480 Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala ThrGly 485 490 495 Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr His ArgSer Ala 500 505 510 Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile Thr GlnIle Pro Ala 515 520 525 Val Lys Cys Trp Asp Asn Leu Pro Phe Val Pro ValVal Lys Gly Pro 530 535 540 Gly His Thr Gly Gly Asp Leu Leu Gln Tyr AsnArg Ser Thr Gly Ser 545 550 555 560 Val Gly Thr Leu Phe Leu Ala Arg TyrGly Leu Ala Leu Glu Lys Ala 565 570 575 Gly Lys Tyr Arg Val Arg Leu ArgTyr Ala Thr Asp Ala Asp Ile Val 580 585 590 Leu His Val Asn Asp Ala GlnIle Gln Met Pro Lys Thr Met Asn Pro 595 600 605 Gly Glu Asp Leu Thr SerLys Thr Phe Lys Val Ala Asp Ala Ile Thr 610 615 620 Thr Leu Asn Leu AlaThr Asp Ser Ser Leu Ala Leu Lys His Asn Leu 625 630 635 640 Gly Glu AspPro Asn Ser Thr Leu Ser Gly Ile Val Tyr Val Asp Arg 645 650 655 Ile GluPhe Ile Pro Val Asp Glu Thr Tyr Glu Ala Glu 660 665 <210> SEQ ID NO 17<211> LENGTH: 2022 <212> TYPE: DNA <213> ORGANISM: Bacillusthuringiensis (truncated) <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)...(2022) <221> NAME/KEY: misc_feature <222> LOCATION:(0)...(0) <223> OTHER INFORMATION: 1218-2A <400> SEQUENCE: 17 atg agtcca aat aat caa aat gaa tat gaa att ata gat gcg aca cct 48 Met Ser ProAsn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 tct acttct gta tcc aat gat tct aac aga tac cct ttt gcg aat gag 96 Ser Thr SerVal Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 cca aca aatgcg cta caa aat atg gat tat aaa gat tat tta aaa atg 144 Pro Thr Asn AlaLeu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45 tct gcg gga aatgct agt gaa tac cct ggt tca cct gaa gta ctt gtt 192 Ser Ala Gly Asn AlaSer Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 50 55 60 agc gga caa gat gcagct aag gcc gca att gat ata gta ggt aaa tta 240 Ser Gly Gln Asp Ala AlaLys Ala Ala Ile Asp Ile Val Gly Lys Leu 65 70 75 80 cta tca ggt tta ggggtc cca ttt gtt ggg ccg ata gtg agt ctt tat 288 Leu Ser Gly Leu Gly ValPro Phe Val Gly Pro Ile Val Ser Leu Tyr 85 90 95 act caa ctt att gat attctg tgg cct tca ggg caa aag agt caa tgg 336 Thr Gln Leu Ile Asp Ile LeuTrp Pro Ser Gly Gln Lys Ser Gln Trp 100 105 110 gag att ttt atg gaa caagta gaa gaa ctc ata aat caa aaa ata gca 384 Glu Ile Phe Met Glu Gln ValGlu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 gaa tat gca agg aat aaagcg ctt tcg gaa tta gaa gga tta ggt aat 432 Glu Tyr Ala Arg Asn Lys AlaLeu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 aat tac caa tta tat ctaact gcg ctt gaa gaa tgg aaa gaa aat cca 480 Asn Tyr Gln Leu Tyr Leu ThrAla Leu Glu Glu Trp Lys Glu Asn Pro 145 150 155 160 aat ggt tca aga gcctta cga gat gtg cga aat cga ttt gaa atc ctg 528 Asn Gly Ser Arg Ala LeuArg Asp Val Arg Asn Arg Phe Glu Ile Leu 165 170 175 gat agt tta ttt acgcaa tac atg cca tct ttt cga gtg aca aat ttt 576 Asp Ser Leu Phe Thr GlnTyr Met Pro Ser Phe Arg Val Thr Asn Phe 180 185 190 gaa gta cca ttc cttaca gta tat aca cag gca gcc aac ctt cat tta 624 Glu Val Pro Phe Leu ThrVal Tyr Thr Gln Ala Ala Asn Leu His Leu 195 200 205 ctg tta tta aag gacgct tca att ttt gga gaa gaa tgg gga tgg tct 672 Leu Leu Leu Lys Asp AlaSer Ile Phe Gly Glu Glu Trp Gly Trp Ser 210 215 220 aca acc act att aataac tat tat gat cgt caa atg aaa ctt act gca 720 Thr Thr Thr Ile Asn AsnTyr Tyr Asp Arg Gln Met Lys Leu Thr Ala 225 230 235 240 gaa tat tct gatcac tgt gta aag tgg tat gaa act ggt tta gca aaa 768 Glu Tyr Ser Asp HisCys Val Lys Trp Tyr Glu Thr Gly Leu Ala Lys 245 250 255 tta aaa ggc acgagc gct aaa caa tgg gtc gac tat aac caa ttc cgt 816 Leu Lys Gly Thr SerAla Lys Gln Trp Val Asp Tyr Asn Gln Phe Arg 260 265 270 aga gaa atg acactg acg gtt tta gat gtt gtt gca tta ttc cca aat 864 Arg Glu Met Thr LeuThr Val Leu Asp Val Val Ala Leu Phe Pro Asn 275 280 285 tat gac aca cgcacg tac cca atg gaa acg aaa gca caa cta aca agg 912 Tyr Asp Thr Arg ThrTyr Pro Met Glu Thr Lys Ala Gln Leu Thr Arg 290 295 300 gaa gta tat acagat cca ctg ggc gcg gta aac gtg tct tca att ggt 960 Glu Val Tyr Thr AspPro Leu Gly Ala Val Asn Val Ser Ser Ile Gly 305 310 315 320 tcc tgg tatgac aaa gca cct tct ttc gga gtg ata gaa tca tcc gtt 1008 Ser Trp Tyr AspLys Ala Pro Ser Phe Gly Val Ile Glu Ser Ser Val 325 330 335 att cga ccaccc cat gta ttt gat tat ata acg gga ctc aca gtg tat 1056 Ile Arg Pro ProHis Val Phe Asp Tyr Ile Thr Gly Leu Thr Val Tyr 340 345 350 aca caa tcaaga agc att tct tcc gct cgc tat ata aga cat tgg gct 1104 Thr Gln Ser ArgSer Ile Ser Ser Ala Arg Tyr Ile Arg His Trp Ala 355 360 365 ggt cat caaata agc tat cat cgg att ttt agt gat aat att ata aaa 1152 Gly His Gln IleSer Tyr His Arg Ile Phe Ser Asp Asn Ile Ile Lys 370 375 380 cag atg tatgga act aat caa aat cta cac agc act agt acc ttt gat 1200 Gln Met Tyr GlyThr Asn Gln Asn Leu His Ser Thr Ser Thr Phe Asp 385 390 395 400 ttt acgaat tat gat att tac aag acg tta tca aaa gat gcg gtg ctc 1248 Phe Thr AsnTyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val Leu 405 410 415 ctt gatatt gtt ttt cct ggt tat acg tat ata ttt ttt gga atg cca 1296 Leu Asp IleVal Phe Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met Pro 420 425 430 gaa gtcgag ttt ttc atg gta aac caa ttg aat aat acc aga aag acg 1344 Glu Val GluPhe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys Thr 435 440 445 tta aagtat aat ccg gtt tcc aaa gat att ata gcg ggg aca aga gat 1392 Leu Lys TyrAsn Pro Val Ser Lys Asp Ile Ile Ala Gly Thr Arg Asp 450 455 460 tcg gaatta gaa tta cct cca gaa act tca gat caa cca aat tat gag 1440 Ser Glu LeuGlu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr Glu 465 470 475 480 tcatat agc cat aga tta tgt cat atc aca agt att ccc gcg acg ggt 1488 Ser TyrSer His Arg Leu Cys His Ile Thr Ser Ile Pro Ala Thr Gly 485 490 495 tcaact acc gga tta gta cct gta ttt tct tgg aca cat cgg agt gcc 1536 Ser ThrThr Gly Leu Val Pro Val Phe Ser Trp Thr His Arg Ser Ala 500 505 510 gatctt ata aat gca gtt cat tca gat aaa att act cag att ccg gtc 1584 Asp LeuIle Asn Ala Val His Ser Asp Lys Ile Thr Gln Ile Pro Val 515 520 525 gtaaag gtt tct gat ttg gct ccc tct ata aca gga ggg cca aat aat 1632 Val LysVal Ser Asp Leu Ala Pro Ser Ile Thr Gly Gly Pro Asn Asn 530 535 540 accgtt gta tcg ggt cct gga ttt aca ggg ggg ggg ata ata aaa gta 1680 Thr ValVal Ser Gly Pro Gly Phe Thr Gly Gly Gly Ile Ile Lys Val 545 550 555 560ata aga aat gga gta att ata tca cat atg cgt gtt aaa att tca gac 1728 IleArg Asn Gly Val Ile Ile Ser His Met Arg Val Lys Ile Ser Asp 565 570 575att aac aaa gaa tat agt atg agg att cgg tat gct tcc gct aat aat 1776 IleAsn Lys Glu Tyr Ser Met Arg Ile Arg Tyr Ala Ser Ala Asn Asn 580 585 590act gaa ttt tat ata aat cct tct gaa gaa aac gtt aaa tct cac gct 1824 ThrGlu Phe Tyr Ile Asn Pro Ser Glu Glu Asn Val Lys Ser His Ala 595 600 605caa aaa act atg aat aga ggt gaa gct tta aca tat aat aaa ttt aat 1872 GlnLys Thr Met Asn Arg Gly Glu Ala Leu Thr Tyr Asn Lys Phe Asn 610 615 620tat gcg act ttg ccc cct att aaa ttt acg aca acc gaa cct ttc att 1920 TyrAla Thr Leu Pro Pro Ile Lys Phe Thr Thr Thr Glu Pro Phe Ile 625 630 635640 act cta ggg gct ata ttt gaa gcg gaa gac ttt ctt gga att gaa gct 1968Thr Leu Gly Ala Ile Phe Glu Ala Glu Asp Phe Leu Gly Ile Glu Ala 645 650655 tat ata gac cga atc gaa ttt atc cca gta gat gag aca tat gaa gcg 2016Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Asp Glu Thr Tyr Glu Ala 660 665670 gaa taa 2022 Glu * <210> SEQ ID NO 18 <211> LENGTH: 673 <212> TYPE:PRT <213> ORGANISM: Bacillus thuringiensis (truncated) <400> SEQUENCE:18 Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 1 510 15 Ser Thr Ser Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 2025 30 Pro Thr Asn Ala Leu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 3540 45 Ser Ala Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 5055 60 Ser Gly Gln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly Lys Leu 6570 75 80 Leu Ser Gly Leu Gly Val Pro Phe Val Gly Pro Ile Val Ser Leu Tyr85 90 95 Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly Gln Lys Ser Gln Trp100 105 110 Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile Asn Gln Lys IleAla 115 120 125 Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu Glu Gly LeuGly Asn 130 135 140 Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu Trp LysGlu Asn Pro 145 150 155 160 Asn Gly Ser Arg Ala Leu Arg Asp Val Arg AsnArg Phe Glu Ile Leu 165 170 175 Asp Ser Leu Phe Thr Gln Tyr Met Pro SerPhe Arg Val Thr Asn Phe 180 185 190 Glu Val Pro Phe Leu Thr Val Tyr ThrGln Ala Ala Asn Leu His Leu 195 200 205 Leu Leu Leu Lys Asp Ala Ser IlePhe Gly Glu Glu Trp Gly Trp Ser 210 215 220 Thr Thr Thr Ile Asn Asn TyrTyr Asp Arg Gln Met Lys Leu Thr Ala 225 230 235 240 Glu Tyr Ser Asp HisCys Val Lys Trp Tyr Glu Thr Gly Leu Ala Lys 245 250 255 Leu Lys Gly ThrSer Ala Lys Gln Trp Val Asp Tyr Asn Gln Phe Arg 260 265 270 Arg Glu MetThr Leu Thr Val Leu Asp Val Val Ala Leu Phe Pro Asn 275 280 285 Tyr AspThr Arg Thr Tyr Pro Met Glu Thr Lys Ala Gln Leu Thr Arg 290 295 300 GluVal Tyr Thr Asp Pro Leu Gly Ala Val Asn Val Ser Ser Ile Gly 305 310 315320 Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val Ile Glu Ser Ser Val 325330 335 Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr Gly Leu Thr Val Tyr340 345 350 Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr Ile Arg His TrpAla 355 360 365 Gly His Gln Ile Ser Tyr His Arg Ile Phe Ser Asp Asn IleIle Lys 370 375 380 Gln Met Tyr Gly Thr Asn Gln Asn Leu His Ser Thr SerThr Phe Asp 385 390 395 400 Phe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu SerLys Asp Ala Val Leu 405 410 415 Leu Asp Ile Val Phe Pro Gly Tyr Thr TyrIle Phe Phe Gly Met Pro 420 425 430 Glu Val Glu Phe Phe Met Val Asn GlnLeu Asn Asn Thr Arg Lys Thr 435 440 445 Leu Lys Tyr Asn Pro Val Ser LysAsp Ile Ile Ala Gly Thr Arg Asp 450 455 460 Ser Glu Leu Glu Leu Pro ProGlu Thr Ser Asp Gln Pro Asn Tyr Glu 465 470 475 480 Ser Tyr Ser His ArgLeu Cys His Ile Thr Ser Ile Pro Ala Thr Gly 485 490 495 Ser Thr Thr GlyLeu Val Pro Val Phe Ser Trp Thr His Arg Ser Ala 500 505 510 Asp Leu IleAsn Ala Val His Ser Asp Lys Ile Thr Gln Ile Pro Val 515 520 525 Val LysVal Ser Asp Leu Ala Pro Ser Ile Thr Gly Gly Pro Asn Asn 530 535 540 ThrVal Val Ser Gly Pro Gly Phe Thr Gly Gly Gly Ile Ile Lys Val 545 550 555560 Ile Arg Asn Gly Val Ile Ile Ser His Met Arg Val Lys Ile Ser Asp 565570 575 Ile Asn Lys Glu Tyr Ser Met Arg Ile Arg Tyr Ala Ser Ala Asn Asn580 585 590 Thr Glu Phe Tyr Ile Asn Pro Ser Glu Glu Asn Val Lys Ser HisAla 595 600 605 Gln Lys Thr Met Asn Arg Gly Glu Ala Leu Thr Tyr Asn LysPhe Asn 610 615 620 Tyr Ala Thr Leu Pro Pro Ile Lys Phe Thr Thr Thr GluPro Phe Ile 625 630 635 640 Thr Leu Gly Ala Ile Phe Glu Ala Glu Asp PheLeu Gly Ile Glu Ala 645 650 655 Tyr Ile Asp Arg Ile Glu Phe Ile Pro ValAsp Glu Thr Tyr Glu Ala 660 665 670 Glu <210> SEQ ID NO 19 <211> LENGTH:1860 <212> TYPE: DNA <213> ORGANISM: Bacillus thuringiensis (truncated)<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (10)...(1860) <221>NAME/KEY: misc_feature <222> LOCATION: (0)...(0) <223> OTHERINFORMATION: 49PVD <400> SEQUENCE: 19 tccatgggc atg tct gcg gga aat gctagt gaa tac cct ggt tca cct gaa 51 Met Ser Ala Gly Asn Ala Ser Glu TyrPro Gly Ser Pro Glu 1 5 10 gta ctt gtt agc gga caa gat gca gct aag gccgca att gat ata gta 99 Val Leu Val Ser Gly Gln Asp Ala Ala Lys Ala AlaIle Asp Ile Val 15 20 25 30 ggt aaa tta cta tca ggt tta ggg gtc cca tttgtt ggg ccg ata gtg 147 Gly Lys Leu Leu Ser Gly Leu Gly Val Pro Phe ValGly Pro Ile Val 35 40 45 agt ctt tat act caa ctt att gat att ctg tgg ccttca ggg gaa aag 195 Ser Leu Tyr Thr Gln Leu Ile Asp Ile Leu Trp Pro SerGly Glu Lys 50 55 60 agt caa tgg gaa att ttt atg gaa caa gta gaa gaa ctcatt aat caa 243 Ser Gln Trp Glu Ile Phe Met Glu Gln Val Glu Glu Leu IleAsn Gln 65 70 75 aaa ata gca gaa tat gca agg aat aaa gcg ctt tcg gaa ttagaa gga 291 Lys Ile Ala Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu GluGly 80 85 90 tta ggt aat aat tac caa tta tat cta act gcg ctt gaa gaa tgggaa 339 Leu Gly Asn Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu Trp Glu95 100 105 110 gaa aat cca aat ggt tca aga gcc tta cga gat gtg cga aatcga ttt 387 Glu Asn Pro Asn Gly Ser Arg Ala Leu Arg Asp Val Arg Asn ArgPhe 115 120 125 gaa atc ctg gat agt tta ttt acg caa tat atg cca tct tttaga gtg 435 Glu Ile Leu Asp Ser Leu Phe Thr Gln Tyr Met Pro Ser Phe ArgVal 130 135 140 aca aat ttt gaa gta cca ttc ctt act gta tat gca atg gcagcc aac 483 Thr Asn Phe Glu Val Pro Phe Leu Thr Val Tyr Ala Met Ala AlaAsn 145 150 155 ctt cat tta ctg tta tta aag gac gcg tca att ttt gga gaagaa tgg 531 Leu His Leu Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu GluTrp 160 165 170 gga tgg tca aca act act att aat aac tat tat gat cgt caaatg aaa 579 Gly Trp Ser Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln MetLys 175 180 185 190 ctt act gca gaa tat tct gat cac tgt gta aag tgg tatgaa act ggt 627 Leu Thr Ala Glu Tyr Ser Asp His Cys Val Lys Trp Tyr GluThr Gly 195 200 205 tta gca aaa tta aaa ggc acg agc gct aaa caa tgg gttgac tat aac 675 Leu Ala Lys Leu Lys Gly Thr Ser Ala Lys Gln Trp Val AspTyr Asn 210 215 220 caa ttc cgt aga gaa atg aca ctg gcg gtt tta gat gttgtt gca tta 723 Gln Phe Arg Arg Glu Met Thr Leu Ala Val Leu Asp Val ValAla Leu 225 230 235 ttc cca aat tat gac aca cgc acg tac cca atg gaa acgaaa gca caa 771 Phe Pro Asn Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr LysAla Gln 240 245 250 cta aca agg gaa gta tat aca gat cca ctg ggc gcg gtaaac gtg tct 819 Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Gly Ala Val AsnVal Ser 255 260 265 270 tca att ggt tcc tgg tat gac aaa gca cct tct ttcgga gtg ata gaa 867 Ser Ile Gly Ser Trp Tyr Asp Lys Ala Pro Ser Phe GlyVal Ile Glu 275 280 285 tca tcc gtt att cga cca ccc cat gta ttt gat tatata acg gga ctc 915 Ser Ser Val Ile Arg Pro Pro His Val Phe Asp Tyr IleThr Gly Leu 290 295 300 aca gtg tat aca caa tca aga agc att tct tcc gctcgc tat ata aga 963 Thr Val Tyr Thr Gln Ser Arg Ser Ile Ser Ser Ala ArgTyr Ile Arg 305 310 315 cat tgg gct ggt cat caa ata agc tac cat cgt gtcagt agg ggt agt 1011 His Trp Ala Gly His Gln Ile Ser Tyr His Arg Val SerArg Gly Ser 320 325 330 aat ctt caa caa atg tat gga act aat caa aat ctacac agc act agt 1059 Asn Leu Gln Gln Met Tyr Gly Thr Asn Gln Asn Leu HisSer Thr Ser 335 340 345 350 acc ttt gat ttt acg aat tat gat att tac aagact cta tca aag gat 1107 Thr Phe Asp Phe Thr Asn Tyr Asp Ile Tyr Lys ThrLeu Ser Lys Asp 355 360 365 gca gta ctc ctt gat att gtt tac cct ggt tatacg tat ata ttt ttt 1155 Ala Val Leu Leu Asp Ile Val Tyr Pro Gly Tyr ThrTyr Ile Phe Phe 370 375 380 gga atg cca gaa gtc gag ttt ttc atg gta aaccaa ttg aat aat acc 1203 Gly Met Pro Glu Val Glu Phe Phe Met Val Asn GlnLeu Asn Asn Thr 385 390 395 aga aag acg tta aag tat aat cca gtt tcc aaagat att ata gcg agt 1251 Arg Lys Thr Leu Lys Tyr Asn Pro Val Ser Lys AspIle Ile Ala Ser 400 405 410 aca aga gat tcg gaa tta gaa tta cct cca gaaact tca gat caa cca 1299 Thr Arg Asp Ser Glu Leu Glu Leu Pro Pro Glu ThrSer Asp Gln Pro 415 420 425 430 aat tat gag tca tat agc cat aga tta tgtcat atc aca agt att ccc 1347 Asn Tyr Glu Ser Tyr Ser His Arg Leu Cys HisIle Thr Ser Ile Pro 435 440 445 gcg acg ggt aac act acc gga tta gta cctgta ttt tct tgg aca cat 1395 Ala Thr Gly Asn Thr Thr Gly Leu Val Pro ValPhe Ser Trp Thr His 450 455 460 cga agt gca gat tta aac aat aca ata tattca gat aaa atc act caa 1443 Arg Ser Ala Asp Leu Asn Asn Thr Ile Tyr SerAsp Lys Ile Thr Gln 465 470 475 att ccg gcc gtt aaa tgt tgg gat aat ttaccg ttt gtt cca gtg gta 1491 Ile Pro Ala Val Lys Cys Trp Asp Asn Leu ProPhe Val Pro Val Val 480 485 490 aaa gga cca gga cat aca gga ggg gat ttatta cag tat aat aga agt 1539 Lys Gly Pro Gly His Thr Gly Gly Asp Leu LeuGln Tyr Asn Arg Ser 495 500 505 510 act ggt tct gta gga acc tta ttt ctagct cga tat ggc cta gca tta 1587 Thr Gly Ser Val Gly Thr Leu Phe Leu AlaArg Tyr Gly Leu Ala Leu 515 520 525 gaa aaa gca ggg aaa tat cgt gta agactg aga tat gct act gat gca 1635 Glu Lys Ala Gly Lys Tyr Arg Val Arg LeuArg Tyr Ala Thr Asp Ala 530 535 540 gat att gta ttg cat gta aac gat gctcag att cag atg cca aaa aca 1683 Asp Ile Val Leu His Val Asn Asp Ala GlnIle Gln Met Pro Lys Thr 545 550 555 atg aac cca ggt gag gat ctg aca tctaaa act ttt aaa gtt gca gat 1731 Met Asn Pro Gly Glu Asp Leu Thr Ser LysThr Phe Lys Val Ala Asp 560 565 570 gct atc aca aca tta aat tta gca acagat agt tcg cta gca ttg aaa 1779 Ala Ile Thr Thr Leu Asn Leu Ala Thr AspSer Ser Leu Ala Leu Lys 575 580 585 590 cat aat tta ggt gaa gac cct aattca aca tta tct ggt ata gtt tac 1827 His Asn Leu Gly Glu Asp Pro Asn SerThr Leu Ser Gly Ile Val Tyr 595 600 605 gtt gac cga atc gaa ttc atc ccagta gat taa 1860 Val Asp Arg Ile Glu Phe Ile Pro Val Asp * 610 615 <210>SEQ ID NO 20 <211> LENGTH: 616 <212> TYPE: PRT <213> ORGANISM: Bacillusthuringiensis (truncated) <400> SEQUENCE: 20 Met Ser Ala Gly Asn Ala SerGlu Tyr Pro Gly Ser Pro Glu Val Leu 1 5 10 15 Val Ser Gly Gln Asp AlaAla Lys Ala Ala Ile Asp Ile Val Gly Lys 20 25 30 Leu Leu Ser Gly Leu GlyVal Pro Phe Val Gly Pro Ile Val Ser Leu 35 40 45 Tyr Thr Gln Leu Ile AspIle Leu Trp Pro Ser Gly Glu Lys Ser Gln 50 55 60 Trp Glu Ile Phe Met GluGln Val Glu Glu Leu Ile Asn Gln Lys Ile 65 70 75 80 Ala Glu Tyr Ala ArgAsn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly 85 90 95 Asn Asn Tyr Gln LeuTyr Leu Thr Ala Leu Glu Glu Trp Glu Glu Asn 100 105 110 Pro Asn Gly SerArg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu Ile 115 120 125 Leu Asp SerLeu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn 130 135 140 Phe GluVal Pro Phe Leu Thr Val Tyr Ala Met Ala Ala Asn Leu His 145 150 155 160Leu Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp 165 170175 Ser Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu Thr 180185 190 Ala Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu Ala195 200 205 Lys Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr Asn GlnPhe 210 215 220 Arg Arg Glu Met Thr Leu Ala Val Leu Asp Val Val Ala LeuPhe Pro 225 230 235 240 Asn Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr LysAla Gln Leu Thr 245 250 255 Arg Glu Val Tyr Thr Asp Pro Leu Gly Ala ValAsn Val Ser Ser Ile 260 265 270 Gly Ser Trp Tyr Asp Lys Ala Pro Ser PheGly Val Ile Glu Ser Ser 275 280 285 Val Ile Arg Pro Pro His Val Phe AspTyr Ile Thr Gly Leu Thr Val 290 295 300 Tyr Thr Gln Ser Arg Ser Ile SerSer Ala Arg Tyr Ile Arg His Trp 305 310 315 320 Ala Gly His Gln Ile SerTyr His Arg Val Ser Arg Gly Ser Asn Leu 325 330 335 Gln Gln Met Tyr GlyThr Asn Gln Asn Leu His Ser Thr Ser Thr Phe 340 345 350 Asp Phe Thr AsnTyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val 355 360 365 Leu Leu AspIle Val Tyr Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met 370 375 380 Pro GluVal Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys 385 390 395 400Thr Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Ser Thr Arg 405 410415 Asp Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr 420425 430 Glu Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala Thr435 440 445 Gly Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr His ArgSer 450 455 460 Ala Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile Thr GlnIle Pro 465 470 475 480 Ala Val Lys Cys Trp Asp Asn Leu Pro Phe Val ProVal Val Lys Gly 485 490 495 Pro Gly His Thr Gly Gly Asp Leu Leu Gln TyrAsn Arg Ser Thr Gly 500 505 510 Ser Val Gly Thr Leu Phe Leu Ala Arg TyrGly Leu Ala Leu Glu Lys 515 520 525 Ala Gly Lys Tyr Arg Val Arg Leu ArgTyr Ala Thr Asp Ala Asp Ile 530 535 540 Val Leu His Val Asn Asp Ala GlnIle Gln Met Pro Lys Thr Met Asn 545 550 555 560 Pro Gly Glu Asp Leu ThrSer Lys Thr Phe Lys Val Ala Asp Ala Ile 565 570 575 Thr Thr Leu Asn LeuAla Thr Asp Ser Ser Leu Ala Leu Lys His Asn 580 585 590 Leu Gly Glu AspPro Asn Ser Thr Leu Ser Gly Ile Val Tyr Val Asp 595 600 605 Arg Ile GluPhe Ile Pro Val Asp 610 615 <210> SEQ ID NO 21 <211> LENGTH: 2022 <212>TYPE: DNA <213> ORGANISM: Bacillus thuringiensis (mutated) <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(2022) <221>NAME/KEY: misc_feature <222> LOCATION: (0)...(0) <223> OTHERINFORMATION: LKMS.N1218-1 <400> SEQUENCE: 21 atg agt cca aat aat caa aatgaa tat gaa att ata gat gcg aca cct 48 Met Ser Pro Asn Asn Gln Asn GluTyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 tct act tct gta tcc aat gattct aac aga tac cct ttt gcg aat gag 96 Ser Thr Ser Val Ser Asn Asp SerAsn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 cca aca aat gcg cta caa aat atggat tat aaa gat tat tta aaa atg 144 Pro Thr Asn Ala Leu Gln Asn Met AspTyr Lys Asp Tyr Leu Lys Met 35 40 45 tct gcg gga aat gct agt gaa tac cctggt tca cct gaa gta ctt gtt 192 Ser Ala Gly Asn Ala Ser Glu Tyr Pro GlySer Pro Glu Val Leu Val 50 55 60 agc gga caa gat gca gct aag gcc gca attgat ata gta ggt aaa tta 240 Ser Gly Gln Asp Ala Ala Lys Ala Ala Ile AspIle Val Gly Lys Leu 65 70 75 80 cta tca ggt tta ggg gtc cca ttt gtt gggccg ata gtg agt ctt tat 288 Leu Ser Gly Leu Gly Val Pro Phe Val Gly ProIle Val Ser Leu Tyr 85 90 95 act caa ctt att gat att ctg tgg cct tca ggggaa aag agt caa tgg 336 Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly GluLys Ser Gln Trp 100 105 110 gaa att ttt atg gaa caa gta gaa gaa ctc attaat caa aaa ata gca 384 Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile AsnGln Lys Ile Ala 115 120 125 gaa tat gca agg aat aaa gcg ctt tcg gaa ttagaa gga tta ggt aat 432 Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu GluGly Leu Gly Asn 130 135 140 aat tac caa tta tat cta act gcg ctt gaa gaatgg gaa gaa aat cca 480 Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu TrpGlu Glu Asn Pro 145 150 155 160 tta aaa atg tct aat ggt tca aga gcc ttacga gat gtg cga aat cga 528 Leu Lys Met Ser Asn Gly Ser Arg Ala Leu ArgAsp Val Arg Asn Arg 165 170 175 ttt gaa atc ctg gat agt tta ttt acg caatat atg cca tct ttt aga 576 Phe Glu Ile Leu Asp Ser Leu Phe Thr Gln TyrMet Pro Ser Phe Arg 180 185 190 gtg aca aat ttt gaa gta cca ttc ctt actgta tat gca atg gca gcc 624 Val Thr Asn Phe Glu Val Pro Phe Leu Thr ValTyr Ala Met Ala Ala 195 200 205 aac ctt cat tta ctg tta tta aag gac gcgtca att ttt gga gaa gaa 672 Asn Leu His Leu Leu Leu Leu Lys Asp Ala SerIle Phe Gly Glu Glu 210 215 220 tgg gga tgg tca aca act act att aat aactat tat gat cgt caa atg 720 Trp Gly Trp Ser Thr Thr Thr Ile Asn Asn TyrTyr Asp Arg Gln Met 225 230 235 240 aaa ctt act gca gaa tat tct gat cactgt gta aag tgg tat gaa act 768 Lys Leu Thr Ala Glu Tyr Ser Asp His CysVal Lys Trp Tyr Glu Thr 245 250 255 ggt tta gca aaa tta aaa ggc acg agcgct aaa caa tgg gtt gac tat 816 Gly Leu Ala Lys Leu Lys Gly Thr Ser AlaLys Gln Trp Val Asp Tyr 260 265 270 aac caa ttc cgt aga gaa atg aca ctggcg gtt tta gat gtt gtt gca 864 Asn Gln Phe Arg Arg Glu Met Thr Leu AlaVal Leu Asp Val Val Ala 275 280 285 tta ttc cca aat tat gac aca cgc acgtac cca atg gaa acg aaa gca 912 Leu Phe Pro Asn Tyr Asp Thr Arg Thr TyrPro Met Glu Thr Lys Ala 290 295 300 caa cta aca agg gaa gta tat aca gatcca ctg ggc gcg gta aac gtg 960 Gln Leu Thr Arg Glu Val Tyr Thr Asp ProLeu Gly Ala Val Asn Val 305 310 315 320 tct tca att ggt tcc tgg tat gacaaa gca cct tct ttc gga gtg ata 1008 Ser Ser Ile Gly Ser Trp Tyr Asp LysAla Pro Ser Phe Gly Val Ile 325 330 335 gaa tca tcc gtt att cga cca ccccat gta ttt gat tat ata acg gga 1056 Glu Ser Ser Val Ile Arg Pro Pro HisVal Phe Asp Tyr Ile Thr Gly 340 345 350 ctc aca gtg tat aca caa tca agaagc att tct tcc gct cgc tat ata 1104 Leu Thr Val Tyr Thr Gln Ser Arg SerIle Ser Ser Ala Arg Tyr Ile 355 360 365 aga cat tgg gct ggt cat caa ataagc tac cat cgt gtc agt agg ggt 1152 Arg His Trp Ala Gly His Gln Ile SerTyr His Arg Val Ser Arg Gly 370 375 380 agt aat ctt caa caa atg tat ggaact aat caa aat cta cac agc act 1200 Ser Asn Leu Gln Gln Met Tyr Gly ThrAsn Gln Asn Leu His Ser Thr 385 390 395 400 agt acc ttt gat ttt acg aattat gat att tac aag act cta tca aag 1248 Ser Thr Phe Asp Phe Thr Asn TyrAsp Ile Tyr Lys Thr Leu Ser Lys 405 410 415 gat gca gta ctc ctt gat attgtt tac cct ggt tat acg tat ata ttt 1296 Asp Ala Val Leu Leu Asp Ile ValTyr Pro Gly Tyr Thr Tyr Ile Phe 420 425 430 ttt gga atg cca gaa gtc gagttt ttc atg gta aac caa ttg aat aat 1344 Phe Gly Met Pro Glu Val Glu PhePhe Met Val Asn Gln Leu Asn Asn 435 440 445 acc aga aag acg tta aag tataat cca gtt tcc aaa gat att ata gcg 1392 Thr Arg Lys Thr Leu Lys Tyr AsnPro Val Ser Lys Asp Ile Ile Ala 450 455 460 agt aca aga gat tcg gaa ttagaa tta cct cca gaa act tca gat caa 1440 Ser Thr Arg Asp Ser Glu Leu GluLeu Pro Pro Glu Thr Ser Asp Gln 465 470 475 480 cca aat tat gag tca tatagc cat aga tta tgt cat atc aca agt att 1488 Pro Asn Tyr Glu Ser Tyr SerHis Arg Leu Cys His Ile Thr Ser Ile 485 490 495 ccc gcg acg ggt aac actacc gga tta gta cct gta ttt tct tgg aca 1536 Pro Ala Thr Gly Asn Thr ThrGly Leu Val Pro Val Phe Ser Trp Thr 500 505 510 cat cga agt gca gat ttaaac aat aca ata tat tca gat aaa atc act 1584 His Arg Ser Ala Asp Leu AsnAsn Thr Ile Tyr Ser Asp Lys Ile Thr 515 520 525 caa att ccg gcc gtt aaatgt tgg gat aat tta ccg ttt gtt cca gtg 1632 Gln Ile Pro Ala Val Lys CysTrp Asp Asn Leu Pro Phe Val Pro Val 530 535 540 gta aaa gga cca gga cataca gga ggg gat tta tta cag tat aat aga 1680 Val Lys Gly Pro Gly His ThrGly Gly Asp Leu Leu Gln Tyr Asn Arg 545 550 555 560 agt act ggt tct gtagga acc tta ttt cta gct cga tat ggc cta gca 1728 Ser Thr Gly Ser Val GlyThr Leu Phe Leu Ala Arg Tyr Gly Leu Ala 565 570 575 tta gaa aaa gca gggaaa tat cgt gta aga ctg aga tat gct act gat 1776 Leu Glu Lys Ala Gly LysTyr Arg Val Arg Leu Arg Tyr Ala Thr Asp 580 585 590 gca gat att gta ttgcat gta aac gat gct cag att cag atg cca aaa 1824 Ala Asp Ile Val Leu HisVal Asn Asp Ala Gln Ile Gln Met Pro Lys 595 600 605 aca atg aac cca ggtgag gat ctg aca tct aaa act ttt aaa gtt gca 1872 Thr Met Asn Pro Gly GluAsp Leu Thr Ser Lys Thr Phe Lys Val Ala 610 615 620 gat gct atc aca acatta aat tta gca aca gat agt tcg cta gca ttg 1920 Asp Ala Ile Thr Thr LeuAsn Leu Ala Thr Asp Ser Ser Leu Ala Leu 625 630 635 640 aaa cat aat ttaggt gaa gac cct aat tca aca tta tct ggt ata gtt 1968 Lys His Asn Leu GlyGlu Asp Pro Asn Ser Thr Leu Ser Gly Ile Val 645 650 655 tac gtt gac cgaatc gaa ttc atc cca gta gat gag aca tat gaa gcg 2016 Tyr Val Asp Arg IleGlu Phe Ile Pro Val Asp Glu Thr Tyr Glu Ala 660 665 670 gaa taa 2022Glu * <210> SEQ ID NO 22 <211> LENGTH: 673 <212> TYPE: PRT <213>ORGANISM: Bacillus thuringiensis (mutated) <400> SEQUENCE: 22 Met SerPro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 SerThr Ser Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 ProThr Asn Ala Leu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45 SerAla Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 50 55 60 SerGly Gln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly Lys Leu 65 70 75 80Leu Ser Gly Leu Gly Val Pro Phe Val Gly Pro Ile Val Ser Leu Tyr 85 90 95Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly Glu Lys Ser Gln Trp 100 105110 Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile Asn Gln Lys Ile Ala 115120 125 Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly Asn130 135 140 Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu Trp Glu Glu AsnPro 145 150 155 160 Leu Lys Met Ser Asn Gly Ser Arg Ala Leu Arg Asp ValArg Asn Arg 165 170 175 Phe Glu Ile Leu Asp Ser Leu Phe Thr Gln Tyr MetPro Ser Phe Arg 180 185 190 Val Thr Asn Phe Glu Val Pro Phe Leu Thr ValTyr Ala Met Ala Ala 195 200 205 Asn Leu His Leu Leu Leu Leu Lys Asp AlaSer Ile Phe Gly Glu Glu 210 215 220 Trp Gly Trp Ser Thr Thr Thr Ile AsnAsn Tyr Tyr Asp Arg Gln Met 225 230 235 240 Lys Leu Thr Ala Glu Tyr SerAsp His Cys Val Lys Trp Tyr Glu Thr 245 250 255 Gly Leu Ala Lys Leu LysGly Thr Ser Ala Lys Gln Trp Val Asp Tyr 260 265 270 Asn Gln Phe Arg ArgGlu Met Thr Leu Ala Val Leu Asp Val Val Ala 275 280 285 Leu Phe Pro AsnTyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys Ala 290 295 300 Gln Leu ThrArg Glu Val Tyr Thr Asp Pro Leu Gly Ala Val Asn Val 305 310 315 320 SerSer Ile Gly Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val Ile 325 330 335Glu Ser Ser Val Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr Gly 340 345350 Leu Thr Val Tyr Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr Ile 355360 365 Arg His Trp Ala Gly His Gln Ile Ser Tyr His Arg Val Ser Arg Gly370 375 380 Ser Asn Leu Gln Gln Met Tyr Gly Thr Asn Gln Asn Leu His SerThr 385 390 395 400 Ser Thr Phe Asp Phe Thr Asn Tyr Asp Ile Tyr Lys ThrLeu Ser Lys 405 410 415 Asp Ala Val Leu Leu Asp Ile Val Tyr Pro Gly TyrThr Tyr Ile Phe 420 425 430 Phe Gly Met Pro Glu Val Glu Phe Phe Met ValAsn Gln Leu Asn Asn 435 440 445 Thr Arg Lys Thr Leu Lys Tyr Asn Pro ValSer Lys Asp Ile Ile Ala 450 455 460 Ser Thr Arg Asp Ser Glu Leu Glu LeuPro Pro Glu Thr Ser Asp Gln 465 470 475 480 Pro Asn Tyr Glu Ser Tyr SerHis Arg Leu Cys His Ile Thr Ser Ile 485 490 495 Pro Ala Thr Gly Asn ThrThr Gly Leu Val Pro Val Phe Ser Trp Thr 500 505 510 His Arg Ser Ala AspLeu Asn Asn Thr Ile Tyr Ser Asp Lys Ile Thr 515 520 525 Gln Ile Pro AlaVal Lys Cys Trp Asp Asn Leu Pro Phe Val Pro Val 530 535 540 Val Lys GlyPro Gly His Thr Gly Gly Asp Leu Leu Gln Tyr Asn Arg 545 550 555 560 SerThr Gly Ser Val Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu Ala 565 570 575Leu Glu Lys Ala Gly Lys Tyr Arg Val Arg Leu Arg Tyr Ala Thr Asp 580 585590 Ala Asp Ile Val Leu His Val Asn Asp Ala Gln Ile Gln Met Pro Lys 595600 605 Thr Met Asn Pro Gly Glu Asp Leu Thr Ser Lys Thr Phe Lys Val Ala610 615 620 Asp Ala Ile Thr Thr Leu Asn Leu Ala Thr Asp Ser Ser Leu AlaLeu 625 630 635 640 Lys His Asn Leu Gly Glu Asp Pro Asn Ser Thr Leu SerGly Ile Val 645 650 655 Tyr Val Asp Arg Ile Glu Phe Ile Pro Val Asp GluThr Tyr Glu Ala 660 665 670 Glu <210> SEQ ID NO 23 <211> LENGTH: 2013<212> TYPE: DNA <213> ORGANISM: Bacillus thuringiensis (mutated) <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(2013) <221>NAME/KEY: misc_feature <222> LOCATION: (0)...(0) <223> OTHERINFORMATION: LKMS.R1218-1 <400> SEQUENCE: 23 atg agt cca aat aat caa aatgaa tat gaa att ata gat gcg aca cct 48 Met Ser Pro Asn Asn Gln Asn GluTyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 tct act tct gta tcc aat gattct aac aga tac cct ttt gcg aat gag 96 Ser Thr Ser Val Ser Asn Asp SerAsn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 cca aca aat gcg cta caa aat atggat tat aaa gat tat tta aaa atg 144 Pro Thr Asn Ala Leu Gln Asn Met AspTyr Lys Asp Tyr Leu Lys Met 35 40 45 tct gcg gga aat gct agt gaa tac cctggt tca cct gaa gta ctt gtt 192 Ser Ala Gly Asn Ala Ser Glu Tyr Pro GlySer Pro Glu Val Leu Val 50 55 60 agc gga caa gat gca gct aag gcc gca attgat ata gta ggt aaa tta 240 Ser Gly Gln Asp Ala Ala Lys Ala Ala Ile AspIle Val Gly Lys Leu 65 70 75 80 cta tca ggt tta ggg gtc cca ttt gtt gggccg ata gtg agt ctt tat 288 Leu Ser Gly Leu Gly Val Pro Phe Val Gly ProIle Val Ser Leu Tyr 85 90 95 act caa ctt att gat att ctg tgg cct tca ggggaa aag agt caa tgg 336 Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly GluLys Ser Gln Trp 100 105 110 gaa att ttt atg gaa caa gta gaa gaa ctc attaat caa aaa ata gca 384 Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile AsnGln Lys Ile Ala 115 120 125 gaa tat gca agg aat aaa gcg ctt tcg gaa ttagaa gga tta ggt aat 432 Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu GluGly Leu Gly Asn 130 135 140 aat tac caa tta tat cta act gcg ctt gaa gaatgg gaa gaa aat cca 480 Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu TrpGlu Glu Asn Pro 145 150 155 160 tta aaa atg tct aga gcc tta cga gat gtgcga aat cga ttt gaa atc 528 Leu Lys Met Ser Arg Ala Leu Arg Asp Val ArgAsn Arg Phe Glu Ile 165 170 175 ctg gat agt tta ttt acg caa tat atg ccatct ttt aga gtg aca aat 576 Leu Asp Ser Leu Phe Thr Gln Tyr Met Pro SerPhe Arg Val Thr Asn 180 185 190 ttt gaa gta cca ttc ctt act gta tat gcaatg gca gcc aac ctt cat 624 Phe Glu Val Pro Phe Leu Thr Val Tyr Ala MetAla Ala Asn Leu His 195 200 205 tta ctg tta tta aag gac gcg tca att tttgga gaa gaa tgg gga tgg 672 Leu Leu Leu Leu Lys Asp Ala Ser Ile Phe GlyGlu Glu Trp Gly Trp 210 215 220 tca aca act act att aat aac tat tat gatcgt caa atg aaa ctt act 720 Ser Thr Thr Thr Ile Asn Asn Tyr Tyr Asp ArgGln Met Lys Leu Thr 225 230 235 240 gca gaa tat tct gat cac tgt gta aagtgg tat gaa act ggt tta gca 768 Ala Glu Tyr Ser Asp His Cys Val Lys TrpTyr Glu Thr Gly Leu Ala 245 250 255 aaa tta aaa ggc acg agc gct aaa caatgg gtt gac tat aac caa ttc 816 Lys Leu Lys Gly Thr Ser Ala Lys Gln TrpVal Asp Tyr Asn Gln Phe 260 265 270 cgt aga gaa atg aca ctg gcg gtt ttagat gtt gtt gca tta ttc cca 864 Arg Arg Glu Met Thr Leu Ala Val Leu AspVal Val Ala Leu Phe Pro 275 280 285 aat tat gac aca cgc acg tac cca atggaa acg aaa gca caa cta aca 912 Asn Tyr Asp Thr Arg Thr Tyr Pro Met GluThr Lys Ala Gln Leu Thr 290 295 300 agg gaa gta tat aca gat cca ctg ggcgcg gta aac gtg tct tca att 960 Arg Glu Val Tyr Thr Asp Pro Leu Gly AlaVal Asn Val Ser Ser Ile 305 310 315 320 ggt tcc tgg tat gac aaa gca ccttct ttc gga gtg ata gaa tca tcc 1008 Gly Ser Trp Tyr Asp Lys Ala Pro SerPhe Gly Val Ile Glu Ser Ser 325 330 335 gtt att cga cca ccc cat gta tttgat tat ata acg gga ctc aca gtg 1056 Val Ile Arg Pro Pro His Val Phe AspTyr Ile Thr Gly Leu Thr Val 340 345 350 tat aca caa tca aga agc att tcttcc gct cgc tat ata aga cat tgg 1104 Tyr Thr Gln Ser Arg Ser Ile Ser SerAla Arg Tyr Ile Arg His Trp 355 360 365 gct ggt cat caa ata agc tac catcgt gtc agt agg ggt agt aat ctt 1152 Ala Gly His Gln Ile Ser Tyr His ArgVal Ser Arg Gly Ser Asn Leu 370 375 380 caa caa atg tat gga act aat caaaat cta cac agc act agt acc ttt 1200 Gln Gln Met Tyr Gly Thr Asn Gln AsnLeu His Ser Thr Ser Thr Phe 385 390 395 400 gat ttt acg aat tat gat atttac aag act cta tca aag gat gca gta 1248 Asp Phe Thr Asn Tyr Asp Ile TyrLys Thr Leu Ser Lys Asp Ala Val 405 410 415 ctc ctt gat att gtt tac cctggt tat acg tat ata ttt ttt gga atg 1296 Leu Leu Asp Ile Val Tyr Pro GlyTyr Thr Tyr Ile Phe Phe Gly Met 420 425 430 cca gaa gtc gag ttt ttc atggta aac caa ttg aat aat acc aga aag 1344 Pro Glu Val Glu Phe Phe Met ValAsn Gln Leu Asn Asn Thr Arg Lys 435 440 445 acg tta aag tat aat cca gtttcc aaa gat att ata gcg agt aca aga 1392 Thr Leu Lys Tyr Asn Pro Val SerLys Asp Ile Ile Ala Ser Thr Arg 450 455 460 gat tcg gaa tta gaa tta cctcca gaa act tca gat caa cca aat tat 1440 Asp Ser Glu Leu Glu Leu Pro ProGlu Thr Ser Asp Gln Pro Asn Tyr 465 470 475 480 gag tca tat agc cat agatta tgt cat atc aca agt att ccc gcg acg 1488 Glu Ser Tyr Ser His Arg LeuCys His Ile Thr Ser Ile Pro Ala Thr 485 490 495 ggt aac act acc gga ttagta cct gta ttt tct tgg aca cat cga agt 1536 Gly Asn Thr Thr Gly Leu ValPro Val Phe Ser Trp Thr His Arg Ser 500 505 510 gca gat tta aac aat acaata tat tca gat aaa atc act caa att ccg 1584 Ala Asp Leu Asn Asn Thr IleTyr Ser Asp Lys Ile Thr Gln Ile Pro 515 520 525 gcc gtt aaa tgt tgg gataat tta ccg ttt gtt cca gtg gta aaa gga 1632 Ala Val Lys Cys Trp Asp AsnLeu Pro Phe Val Pro Val Val Lys Gly 530 535 540 cca gga cat aca gga ggggat tta tta cag tat aat aga agt act ggt 1680 Pro Gly His Thr Gly Gly AspLeu Leu Gln Tyr Asn Arg Ser Thr Gly 545 550 555 560 tct gta gga acc ttattt cta gct cga tat ggc cta gca tta gaa aaa 1728 Ser Val Gly Thr Leu PheLeu Ala Arg Tyr Gly Leu Ala Leu Glu Lys 565 570 575 gca ggg aaa tat cgtgta aga ctg aga tat gct act gat gca gat att 1776 Ala Gly Lys Tyr Arg ValArg Leu Arg Tyr Ala Thr Asp Ala Asp Ile 580 585 590 gta ttg cat gta aacgat gct cag att cag atg cca aaa aca atg aac 1824 Val Leu His Val Asn AspAla Gln Ile Gln Met Pro Lys Thr Met Asn 595 600 605 cca ggt gag gat ctgaca tct aaa act ttt aaa gtt gca gat gct atc 1872 Pro Gly Glu Asp Leu ThrSer Lys Thr Phe Lys Val Ala Asp Ala Ile 610 615 620 aca aca tta aat ttagca aca gat agt tcg cta gca ttg aaa cat aat 1920 Thr Thr Leu Asn Leu AlaThr Asp Ser Ser Leu Ala Leu Lys His Asn 625 630 635 640 tta ggt gaa gaccct aat tca aca tta tct ggt ata gtt tac gtt gac 1968 Leu Gly Glu Asp ProAsn Ser Thr Leu Ser Gly Ile Val Tyr Val Asp 645 650 655 cga atc gaa ttcatc cca gta gat gag aca tat gaa gcg gaa taa 2013 Arg Ile Glu Phe Ile ProVal Asp Glu Thr Tyr Glu Ala Glu * 660 665 670 <210> SEQ ID NO 24 <211>LENGTH: 670 <212> TYPE: PRT <213> ORGANISM: Bacillus thuringiensis(mutated) <400> SEQUENCE: 24 Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu IleIle Asp Ala Thr Pro 1 5 10 15 Ser Thr Ser Val Ser Asn Asp Ser Asn ArgTyr Pro Phe Ala Asn Glu 20 25 30 Pro Thr Asn Ala Leu Gln Asn Met Asp TyrLys Asp Tyr Leu Lys Met 35 40 45 Ser Ala Gly Asn Ala Ser Glu Tyr Pro GlySer Pro Glu Val Leu Val 50 55 60 Ser Gly Gln Asp Ala Ala Lys Ala Ala IleAsp Ile Val Gly Lys Leu 65 70 75 80 Leu Ser Gly Leu Gly Val Pro Phe ValGly Pro Ile Val Ser Leu Tyr 85 90 95 Thr Gln Leu Ile Asp Ile Leu Trp ProSer Gly Glu Lys Ser Gln Trp 100 105 110 Glu Ile Phe Met Glu Gln Val GluGlu Leu Ile Asn Gln Lys Ile Ala 115 120 125 Glu Tyr Ala Arg Asn Lys AlaLeu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 Asn Tyr Gln Leu Tyr LeuThr Ala Leu Glu Glu Trp Glu Glu Asn Pro 145 150 155 160 Leu Lys Met SerArg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu Ile 165 170 175 Leu Asp SerLeu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn 180 185 190 Phe GluVal Pro Phe Leu Thr Val Tyr Ala Met Ala Ala Asn Leu His 195 200 205 LeuLeu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp 210 215 220Ser Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu Thr 225 230235 240 Ala Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu Ala245 250 255 Lys Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr Asn GlnPhe 260 265 270 Arg Arg Glu Met Thr Leu Ala Val Leu Asp Val Val Ala LeuPhe Pro 275 280 285 Asn Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys AlaGln Leu Thr 290 295 300 Arg Glu Val Tyr Thr Asp Pro Leu Gly Ala Val AsnVal Ser Ser Ile 305 310 315 320 Gly Ser Trp Tyr Asp Lys Ala Pro Ser PheGly Val Ile Glu Ser Ser 325 330 335 Val Ile Arg Pro Pro His Val Phe AspTyr Ile Thr Gly Leu Thr Val 340 345 350 Tyr Thr Gln Ser Arg Ser Ile SerSer Ala Arg Tyr Ile Arg His Trp 355 360 365 Ala Gly His Gln Ile Ser TyrHis Arg Val Ser Arg Gly Ser Asn Leu 370 375 380 Gln Gln Met Tyr Gly ThrAsn Gln Asn Leu His Ser Thr Ser Thr Phe 385 390 395 400 Asp Phe Thr AsnTyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val 405 410 415 Leu Leu AspIle Val Tyr Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met 420 425 430 Pro GluVal Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys 435 440 445 ThrLeu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Ser Thr Arg 450 455 460Asp Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr 465 470475 480 Glu Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala Thr485 490 495 Gly Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr His ArgSer 500 505 510 Ala Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile Thr GlnIle Pro 515 520 525 Ala Val Lys Cys Trp Asp Asn Leu Pro Phe Val Pro ValVal Lys Gly 530 535 540 Pro Gly His Thr Gly Gly Asp Leu Leu Gln Tyr AsnArg Ser Thr Gly 545 550 555 560 Ser Val Gly Thr Leu Phe Leu Ala Arg TyrGly Leu Ala Leu Glu Lys 565 570 575 Ala Gly Lys Tyr Arg Val Arg Leu ArgTyr Ala Thr Asp Ala Asp Ile 580 585 590 Val Leu His Val Asn Asp Ala GlnIle Gln Met Pro Lys Thr Met Asn 595 600 605 Pro Gly Glu Asp Leu Thr SerLys Thr Phe Lys Val Ala Asp Ala Ile 610 615 620 Thr Thr Leu Asn Leu AlaThr Asp Ser Ser Leu Ala Leu Lys His Asn 625 630 635 640 Leu Gly Glu AspPro Asn Ser Thr Leu Ser Gly Ile Val Tyr Val Asp 645 650 655 Arg Ile GluPhe Ile Pro Val Asp Glu Thr Tyr Glu Ala Glu 660 665 670 <210> SEQ ID NO25 <211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: LKMS Insert <400> SEQUENCE: 25tta aaa atg tct 12 <210> SEQ ID NO 26 <211> LENGTH: 4 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: LKMS Insert <400> SEQUENCE: 26 Leu Lys Met Ser 1 <210> SEQID NO 27 <211> LENGTH: 4874 <212> TYPE: DNA <213> ORGANISM: Bacillusthuringiensis <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (0)...(0) <223> OTHER INFORMATION: Genomic DNA 1218-1 <400>SEQUENCE: 27 ggtttccatt cccaccggtt ttcactttca aaaaccccaa tacaccgaaacttgtctatg 60 atgtgagtca tttatcttat ggcaaaagag atgtgtaaac gaacgattgacgtagaggat 120 tgtgggcaaa ttgagataga tttacatgtc ttaaaaatta aaggtgttttaccgtttatc 180 gtgaacgtat ccattgaacc gcttagtatg gaacatgtat ataccacaagtggtagagac 240 acatccttat ttttaagttg tcaagaaacg gtatatgtgg atcatattttaaaatatagt 300 gttgatcatg tcccatatta tgtaattgat ggccatcata ttcaagtgcgtgatgtatcg 360 attaaattga tggaagaaaa cccacaaact gctcaaatat cgggtgttttttattttgat 420 tatgcataat tataaaaaat caaaaaatat tttgtgaaga atccctaaaattatcacaac 480 attgtttatt ataaaataac tcatttcaag aaaaatcgta atatttttttatctaacagg 540 aattttatca tctacagaag aatattctta tcatggtaat gaggagggagagtgacagag 600 aggggataga caatcaaaag agtatctaga agagtttgtc atgtaagaacaaaaggaatc 660 tatcgtatat gctactcaaa agaaagtgta aaaaatcttt gtatcttgtatatgtatagg 720 aggaaaatag atgagtccaa ataatcaaaa tgaatatgaa attatagatgcgacaccttc 780 tacttctgta tccaatgatt ctaacagata cccttttgcg aatgagccaacaaatgcgct 840 acaaaatatg gattataaag attatttaaa aatgtctgcg ggaaatgctagtgaataccc 900 tggttcacct gaagtacttg ttagcggaca agatgcagct aaggccgcaattgatatagt 960 aggtaaatta ctatcaggtt taggggtccc atttgttggg ccgatagtgagtctttatac 1020 tcaacttatt gatattctgt ggccttcagg ggaaaagagt caatgggaaatttttatgga 1080 acaagtagaa gaactcatta atcaaaaaat agcagaatat gcaaggaataaagcgctttc 1140 ggaattagaa ggattaggta ataattacca attatatcta actgcgcttgaagaatggga 1200 agaaaatcca aatggttcaa gagccttacg agatgtgcga aatcgatttgaaatcctgga 1260 tagtttattt acgcaatata tgccatcttt tagagtgaca aattttgaagtaccattcct 1320 tactgtatat gcaatggcag ccaaccttca tttactgtta ttaaaggacgcgtcaatttt 1380 tggagaagaa tggggatggt caacaactac tattaataac tattatgatcgtcaaatgaa 1440 acttactgca gaatattctg atcactgtgt aaagtggtat gaaactggtttagcaaaatt 1500 aaaaggcacg agcgctaaac aatgggttga ctataaccaa ttccgtagagaaatgacact 1560 ggcggtttta gatgttgttg cattattccc aaattatgac acacgcacgtacccaatgga 1620 aacgaaagca caactaacaa gggaagtata tacagatcca ctgggcgcggtaaacgtgtc 1680 ttcaattggt tcctggtatg acaaagcacc ttctttcgga gtgatagaatcatccgttat 1740 tcgaccaccc catgtatttg attatataac gggactcaca gtgtatacacaatcaagaag 1800 catttcttcc gctcgctata taagacattg ggctggtcat caaataagctaccatcgtgt 1860 cagtaggggt agtaatcttc aacaaatgta tggaactaat caaaatctacacagcactag 1920 tacctttgat tttacgaatt atgatattta caagactcta tcaaaggatgcagtactcct 1980 tgatattgtt taccctggtt atacgtatat attttttgga atgccagaagtcgagttttt 2040 catggtaaac caattgaata ataccagaaa gacgttaaag tataatccagtttccaaaga 2100 tattatagcg agtacaagag attcggaatt agaattacct ccagaaacttcagatcaacc 2160 aaattatgag tcatatagcc atagattatg tcatatcaca agtattcccgcgacgggtaa 2220 cactaccgga ttagtacctg tattttcttg gacacatcga agtgcagatttaaacaatac 2280 aatatattca gataaaatca ctcaaattcc ggccgttaaa tgttgggataatttaccgtt 2340 tgttccagtg gtaaaaggac caggacatac aggaggggat ttattacagtataatagaag 2400 tactggttct gtaggaacct tatttctagc tcgatatggc ctagcattagaaaaagcagg 2460 gaaatatcgt gtaagactga gatatgctac tgatgcagat attgtattgcatgtaaacga 2520 tgctcagatt cagatgccaa aaacaatgaa cccaggtgag gatctgacatctaaaacttt 2580 taaagttgca gatgctatca caacattaaa tttagcaaca gatagttcgctagcattgaa 2640 acataattta ggtgaagacc ctaattcaac attatctggt atagtttacgttgaccgaat 2700 cgaattcatc ccagtagatg agacatatga agcggaacaa gatttagaagcagcgaagaa 2760 agcagtgaat gccttgttta cgaatacaaa agatggctta cgaccaggcgtaacggatta 2820 tgaagtgaat caagcggcaa acttagtgga atgcctatcg gatgatttgtatccaaatga 2880 aaaacgattg ttatttgatg cagtgagaga ggcaaaacgc ctcagtgaggcacgtaattt 2940 gcttcaagat ccagatttcc aagagataaa tggagaaaat ggctggacggcaagtacggg 3000 aattgaggtt atagaagggg atgctttatt caaagggcgt tatctacgcctaccaggtgc 3060 gagagaaata gatacggaaa cgtatccaac gtatctgtat caaaaagtagaggaaggtgt 3120 attaaaacca tacacaagat atagattgag agggtttgtc ggaagcagtcaaggattgga 3180 aattttcaca attcgtcatc aaacgaaccg aattgtaaaa aatgtaccggatgatttgct 3240 gccagatgta tctcctgtta actcggatgg tagtatcaat cgatgcagcgaacaaaagta 3300 tgtgaatagc cgtttagaag tagaaaaccg ttctggtgaa gcgcatgagttctctattcc 3360 tattgataca ggtgaaatcg attacaatga aaatgcagga atatgggttggatttaagat 3420 tacggaccca gagggatatg caacactcgg aaacctagaa ttggtcgaagagggaccttt 3480 atcaggagac gcattagaac gcttgcaaag agaagaacaa cagtggaagattcaaatgac 3540 aagaagacgt gaagaaacag atagaaggta tatggcatcg aaacaagcggtagatcgttt 3600 atatgccgat tatcaggatc agcaactgaa tcctgatgta gagattacagatcttactgc 3660 ggcccaagat ctgatacagt ccattcctta cgtatataac gaaatgttcccagaaatacc 3720 agggatgaac tatacgaagt ttacagaatt aacagatcga ctccaacaagcgtggagttt 3780 gtatgatcag cgaaatgcca taccaaatgg tgattttcga aatgggttaagtaattggaa 3840 tgcaacgcct ggcgtagaag tacaacaaat caatcataca tctgtccttgtgattccaaa 3900 ctgggatgag caagtttcgc aacagtttac agttcaaccg aatcaaagatatgtgttacg 3960 agttactgcg agaaaagaag gggtaggaaa tggatatgta agtatccgtgatggtggaaa 4020 tcaaacagaa acgcttactt ttagtgcaag cgattatgat acaaatggaatgtataatac 4080 gcaagtgtcc aatacaaatg gatataacac aaataatgcg tataatacacaagcatcgag 4140 tacaaacgga tataacgcaa ataatatgta taatacgcaa gcatcgaatacaaacggata 4200 taacacaaat agtgtgtaca atgatcaaac cggctatatc acaaaaacagtgacattcat 4260 cccgtataca gatcaaatgt ggattgagat gagtgagaca gaaggtacattctatataga 4320 aagtgtagaa ttgattgtag acgtagagta atagtagtac ccctccagatgaaacctgta 4380 tctggagggg ttttttatgc aaaagagtct tttcatacag aatatattggttttacccgg 4440 attacatatt ttgtgaatag gactatggtt ggttacctta cggtacctttttatatccac 4500 cggcattgga aaatgtaaga gggaggataa tcatatatag tcccttccctacacatcaaa 4560 ttccttcgaa agtttctcgt gaatgagagt gaatatttct ttttgtactttattcaggtc 4620 ttgtaagaaa ggaatggtat tcacacaaat gatgggtgtg gatacgtctgttaaacctga 4680 gatatttgta ataatcaagt catagttttt tgcaatctgt ttaaatgagctgagatgtaa 4740 tacatcaatc ttagatagtt gaatcatatg accaaattga tactgcataatattacgaat 4800 aaatagggta tgttccatat ctgaatcaca aaaaatgccg acatgaagaacaggaacctt 4860 ctgttttaaa gctt 4874 <210> SEQ ID NO 28 <211> LENGTH:6613 <212> TYPE: DNA <213> ORGANISM: Bacillus thuringiensis <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (0)...(0) <223>OTHER INFORMATION: Genomic Cry1218-2 <400> SEQUENCE: 28 ttttaggtattcttttaagt tctttataga gacagattaa cgaaaaacta aataagaaat 60 tcaatcccttgatacatgat gcatcggatg ccaaattatt agtacgtatc ttgcgtatat 120 tgtacgaggtcgaattgacg taacagggca cctttttggt caaattgacc aaagaatcca 180 tcctttgcatgagcacttct cgaaaccact tcccatagtg cacttcttat cttttgtata 240 tatttcctaaggatatcgta atccctattt ctgataagag gattttgtca gtgtaggaag 300 agcgaatgtcttttcgtatt tcaaacaaaa aataaaggat gtttatgcac ggaaataatc 360 atcatattaataatgcccag tacataaaga tagatggggg tcattttttg aaatgattcg 420 aaaagactccgttgactcga taggaggtgc acagaaaaat ggaagaaaga tatgcatcgc 480 aagatcagtcggatgtagaa gtttctaatc gcaaggggaa gaaaaaccat acagttccct 540 ttcaatgtatggtttccatt ccaacaggtt ttcaaattca aaaacccaat acaccgaaac 600 ttgtctatgatgtgagtcat ttatctatgg caaaagagat gtgtaaacga acgattgacg 660 tagaggattgtgggcaaatt gagatagatt tacatgtctt aaaaattaaa ggtgttttac 720 cgtttatcgtgaacgtatcc attgaaccgc ttagtatgaa catgtatata ccacaagtgg 780 tagagacacatccttatttt taagttgtca agaaacggta tatgtggatc atattttaaa 840 atatagtgttgatcatgtcc cgtattatgt aattgatggc catcatattc aagtgcgtga 900 tgtatcgattaaattgatgg aagaaaaccc acaaactgct caaatatcgg gtgtttttta 960 ttttgattatgcataatttt aaaaaatcaa aaaatatttt gtgaagaatc cctaaaatta 1020 tcacaacattgtttattata aaataactca tttcaagaaa aatcgtaata tttttttatc 1080 taacaggaattttatcatct acagaagaat attcttatca tggtaatgag gagggagagt 1140 gacagtcaaaagagtacctg gtttgtcgtg taagaaaaaa gaatcgatcg tacaggaaag 1200 ttaaaaaaagtgtaagaaat tttatatctt ttgtatgtat aggaggaaaa tagatgagtc 1260 caaataatcaaaatgaatat gaaattatag atgcgacacc ttctacttct gtatccaatg 1320 attctaacagataccctttt gcgaatgagc caacaaatgc gctacaaaat atggattata 1380 aagattatttaaaaatgtct gcgggaaatg ctagtgaata ccctggttca cctgaagtac 1440 ttgttagcggacaagatgca gctaaggccg caattgatat agtaggtaaa ttactatcag 1500 gtttaggggtcccatttgtt gggccgatag tgagtcttta tactcaactt attgatattc 1560 tgtggccttcagggcaaaag agtcaatggg agatttttat ggaacaagta gaagaactca 1620 taaatcaaaaaatagcagaa tatgcaagga ataaagcgct ttcggaatta gaaggattag 1680 gtaataattaccaattatat ctaactgcgc ttgaagaatg gaaagaaaat ccaaatggtt 1740 caagagccttacgagatgtg cgaaatcgat ttgaaatcct ggatagttta tttacgcaat 1800 acatgccatcttttcgagtg acaaattttg aagtaccatt ccttacagta tatacacagg 1860 cagccaaccttcatttactg ttattaaagg acgcttcaat ttttggagaa gaatggggat 1920 ggtctacaaccactattaat aactattatg atcgtcaaat gaaacttact gcagaatatt 1980 ctgatcactgtgtaaagtgg tatgaaactg gtttagcaaa attaaaaggc acgagcgcta 2040 aacaatgggtcgactataac caattccgta gagaaatgac actgacggtt ttagatgttg 2100 ttgcattattcccaaattat gacacacgca cgtacccaat ggaaacgaaa gcacaactaa 2160 caagggaagtatatacagat ccactgggcg cggtaaacgt gtcttcaatt ggttcctggt 2220 atgacaaagcaccttctttc ggagtgatag aatcatccgt tattcgacca ccccatgtat 2280 ttgattatataacgggactc acagtgtata cacaatcaag aagcatttct tccgctcgct 2340 atataagacattgggctggt catcaaataa gctatcatcg gatttttagt gataatatta 2400 taaaacagatgtatggaact aatcaaaatc tacacagcac tagtaccttt gattttacga 2460 attatgatatttacaagacg ttatcaaaag atgcggtgct ccttgatatt gtttttcctg 2520 gttatacgtatatatttttt ggaatgccag aagtcgagtt tttcatggta aaccaattga 2580 ataataccagaaagacgtta aagtataatc cggtttccaa agatattata gcggggacaa 2640 gagattcggaattagaatta cctccagaaa cttcagatca accaaattat gagtcatata 2700 gccatagattatgtcatatc acaagtattc ccgcgacggg ttcaactacc ggattagtac 2760 ctgtattttcttggacacat cggagtgccg atcttataaa tgcagttcat tcagataaaa 2820 ttactcagattccggtcgta aaggtttctg atttggctcc ctctataaca ggagggccaa 2880 ataataccgttgtatcgggt cctggattta cagggggggg gataataaaa gtaataagaa 2940 atggagtaattatatcacat atgcgtgtta aaatttcaga cattaacaaa gaatatagta 3000 tgaggattcggtatgcttcc gctaataata ctgaatttta tataaatcct tctgaagaaa 3060 acgttaaatctcacgctcaa aaaactatga atagaggtga agctttaaca tataataaat 3120 ttaattatgcgactttgccc cctattaaat ttacgacaac cgaacctttc attactctag 3180 gggctatatttgaagcggaa gactttcttg gaattgaagc ttatatagac cgaatcgaat 3240 ttatcccagtagatgagaca tatgaagcgg aacaagattt agaagcagcg aagaaagcag 3300 tgaatgccttgtttacgaat acaaaagatg gcttacgacc aggcgtaacg gattatgaag 3360 tgaatcaagcggcaaactta gtggaatgcc tatcggatga tttgtatcca aatgaaaaac 3420 gattgttatttgatgcagtg agagaggcaa aacgcctcag tgaggcacgt aatttgcttc 3480 aagatccagatttccaagag ataaatggag aaaatggctg gacggcaagt acgggaattg 3540 aggttatagaaggggatgct ttattcaaag ggcgttatct acgcctacca ggtgcgagag 3600 aaatagatacggaaacgtat ccaacgtatc tgtatcaaaa agtagaggaa ggtgtattaa 3660 aaccatacacaagatataga ttgagagggt ttgtcggaag cagtcaagga ttggaaattt 3720 tcacaattcgtcatcaaacg aaccgaattg taaaaaatgt accggatgat ttgctgccag 3780 atgtatctcctgttaactcg gatggtagta tcaatcgatg cagcgaacaa aagtatgtga 3840 atagccgtttagaagtagaa aaccgttctg gtgaagcgca tgagttctct attcctattg 3900 atacaggtgaaatcgattac aatgaaaatg caggaatatg ggttggattt aagattacgg 3960 acccagagggatatgcaaca ctcggaaacc tagaattggt cgaagaggga cctttatcag 4020 gagacgcattagaacgcttg caaagagaag aacaacagtg gaagattcaa atgacaagaa 4080 gacgtgaagaaacagataga aggtatatgg catcgaaaca agcggtagat cgtttatatg 4140 ccgattatcaggatcagcaa ctgaatcctg atgtagagat tacagatctt actgcggccc 4200 aagatctgatacagtccatt ccttacgtat ataacgaaat gttcccagaa ataccaggga 4260 tgaactatacgaagtttaca gaattaacag atcgactcca acaagcgtgg agtttgtatg 4320 atcagcgaaatgccatacca aatggtgatt ttcgaaatgg gttaagtaat tggaatgcaa 4380 cgcctggcgtagaagtacaa caaatcaatc atacatctgt ccttgtgatt ccaaactggg 4440 atgagcaagtttcgcaacag tttacagttc aaccgaatca aagatatgtg ttacgagtta 4500 ctgcgagaaaagaaggggta ggaaatggat atgtaagtat ccgtgatggt ggaaatcaaa 4560 cagaaacgcttacttttagt gcaagcgatt atgatacaaa tggaatgtat aatacgcaag 4620 tgtccaatacaaatggatat aacacaaata atgcgtataa tacacaagca tcgagtacaa 4680 acggatataacgcaaataat atgtataata cgcaagcatc gaatacaaac ggatataaca 4740 caaatagtgtgtacaatgat caaaccggct atatcacaaa aacagtgaca ttcatcccgt 4800 atacagatcaaatgtggatt gagatgagtg agacagaagg tacattctat atagaaagtg 4860 tagaattgattgtagacgta gagtaatagt agtacccctc cagatgaaac ctgtatctgg 4920 aggggttttttatgcaaaag agtcttttca tacagaatat attggtttta cccggattac 4980 atattttgtgaataggacta tggttggtta ccttacggta cctttttata tccaccggca 5040 ttggaaaatgtaagagggag gataatcata tatagtccct tccctacaca tcaaattcct 5100 tcgaaagtttctcgtgaatg agagtgaata tttctttttg tactttattc aggtcttgta 5160 agaaaggaatggtattcaca caaatgatgg gtgtggatac gtctgttaaa cctgagatat 5220 ttgtaataatcaagtcatag ttttttgcaa tctgtttaaa tgagctgaga tgtaatacat 5280 caatcttagatagttgaatc atatgaccaa attgatactg cataatatta cgaataaata 5340 gggtatgttccatatctgaa tcacaaaaaa tgccgacatg aagaacagga accttctgtt 5400 ttaaagcttgtaataagttt gtccaatgta tgattaaaat atataatgtt tccgtaaaaa 5460 catgctcgtcccatttgaac tgttcatgat agtgaaagtg agttaattct tcttttaaaa 5520 gcaagacaaagtatgaaaat tcgtgagaat gatgctcgga aaaaaaacgt cttttatcat 5580 gtaaaataaaactacgtcca taattcatgg tttgtaaatt gtataactcc aaaatgattt 5640 tttgtttattttggagaggc acatgtagtt tgtcggatag tctatgcaat aagtttagaa 5700 tttcaggaacaattttccat gcgtcatttg atttttgttg taccatagtt tctaattgct 5760 catacgtaaatgcataatga tgattaaaaa aaacagagaa gagttggtaa acagtctcat 5820 gattaaaatcaagagaaaag gtatcccgga acaattgaca aaatgagctg ttctcaaaaa 5880 tacttacatccaaaggattg gaaaaatctt ctgaaatggt tttcatatgc tggtgttgta 5940 aacgaatcacattcaccatt gtccaatacc gaatccgtat gaggtctgga aaatttagtt 6000 gtatctgatttttttgggtg acatatagaa agagttgatc caatgcctgt agttggttgt 6060 ctgggaaaggagtatgagtc acaccatatt tttcataaaa aaactggacc ataatacttc 6120 taatatgttgttcatttcct atgattttac aaggatttgt ttgaatgtgt atctcatatt 6180 gttcgagatacacattcaat tttgtgataa tccgccttag ggtggaagta ctaagaaata 6240 attcctccgcgattgtctct atgtcatctc tttcatcaaa aaatatccgt tcgataaagg 6300 aaaactcaggacttacagat aagacctttt gatatataaa atcaatagaa tactgagaag 6360 gataggttaacataatccct tttatagatg tctcaatctg aaaaggttga aattcttgat 6420 taataaatttaatgtcatct ctcaaaattc tttcggaaca atttagtgtt tgtgcactca 6480 ctcctaacgtatgccatcca tcttgttcat atagtagttc taagaattgt agttgtctgc 6540 gtaaattgttatttaaaaga gaacgcatga gtagacacct tctttcattt ataaaatatc 6600 actgatggaattc 6613 <210> SEQ ID NO 29 <211> LENGTH: 1863 <212> TYPE: DNA <213>ORGANISM: Bacillus thuringiensis (mutated) <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)...(1863) <221> NAME/KEY: misc_feature<222> LOCATION: (0)...(0) <223> OTHER INFORMATION: NGSR.N49PVD <400>SEQUENCE: 29 atg tct gcg gga aat gct agt gaa tac cct ggt tca cct gaa gtactt 48 Met Ser Ala Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu 15 10 15 gtt agc gga caa gat gca gct aag gcc gca att gat ata gta ggt aaa96 Val Ser Gly Gln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly Lys 20 2530 tta cta tca ggt tta ggg gtc cca ttt gtt ggg ccg ata gtg agt ctt 144Leu Leu Ser Gly Leu Gly Val Pro Phe Val Gly Pro Ile Val Ser Leu 35 40 45tat act caa ctt att gat att ctg tgg cct tca ggg gaa aag agt caa 192 TyrThr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly Glu Lys Ser Gln 50 55 60 tgggaa att ttt atg gaa caa gta gaa gaa ctc att aat caa aaa ata 240 Trp GluIle Phe Met Glu Gln Val Glu Glu Leu Ile Asn Gln Lys Ile 65 70 75 80 gcagaa tat gca agg aat aaa gcg ctt tcg gaa tta gaa gga tta ggt 288 Ala GluTyr Ala Arg Asn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly 85 90 95 aat aattac caa tta tat cta act gcg ctt gaa gaa tgg gaa gaa aat 336 Asn Asn TyrGln Leu Tyr Leu Thr Ala Leu Glu Glu Trp Glu Glu Asn 100 105 110 cca aatggt tca aga aat ggt tcc cgg gcc tta cga gat gtg cga aat 384 Pro Asn GlySer Arg Asn Gly Ser Arg Ala Leu Arg Asp Val Arg Asn 115 120 125 cga tttgaa atc ctg gat agt tta ttt acg caa tat atg cca tct ttt 432 Arg Phe GluIle Leu Asp Ser Leu Phe Thr Gln Tyr Met Pro Ser Phe 130 135 140 aga gtgaca aat ttt gaa gta cca ttc ctt act gta tat gca atg gca 480 Arg Val ThrAsn Phe Glu Val Pro Phe Leu Thr Val Tyr Ala Met Ala 145 150 155 160 gccaac ctt cat tta ctg tta tta aag gac gcg tca att ttt gga gaa 528 Ala AsnLeu His Leu Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu 165 170 175 gaatgg gga tgg tca aca act act att aat aac tat tat gat cgt caa 576 Glu TrpGly Trp Ser Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln 180 185 190 atgaaa ctt act gca gaa tat tct gat cac tgt gta aag tgg tat gaa 624 Met LysLeu Thr Ala Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu 195 200 205 actggt tta gca aaa tta aaa ggc acg agc gct aaa caa tgg gtt gac 672 Thr GlyLeu Ala Lys Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp 210 215 220 tataac caa ttc cgt aga gaa atg aca ctg gcg gtt tta gat gtt gtt 720 Tyr AsnGln Phe Arg Arg Glu Met Thr Leu Ala Val Leu Asp Val Val 225 230 235 240gca tta ttc cca aat tat gac aca cgc acg tac cca atg gaa acg aaa 768 AlaLeu Phe Pro Asn Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys 245 250 255gca caa cta aca agg gaa gta tat aca gat cca ctg ggc gcg gta aac 816 AlaGln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Gly Ala Val Asn 260 265 270gtg tct tca att ggt tcc tgg tat gac aaa gca cct tct ttc gga gtg 864 ValSer Ser Ile Gly Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val 275 280 285ata gaa tca tcc gtt att cga cca ccc cat gta ttt gat tat ata acg 912 IleGlu Ser Ser Val Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr 290 295 300gga ctc aca gtg tat aca caa tca aga agc att tct tcc gct cgc tat 960 GlyLeu Thr Val Tyr Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr 305 310 315320 ata aga cat tgg gct ggt cat caa ata agc tac cat cgt gtc agt agg 1008Ile Arg His Trp Ala Gly His Gln Ile Ser Tyr His Arg Val Ser Arg 325 330335 ggt agt aat ctt caa caa atg tat gga act aat caa aat cta cac agc 1056Gly Ser Asn Leu Gln Gln Met Tyr Gly Thr Asn Gln Asn Leu His Ser 340 345350 act agt acc ttt gat ttt acg aat tat gat att tac aag act cta tca 1104Thr Ser Thr Phe Asp Phe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser 355 360365 aag gat gca gta ctc ctt gat att gtt tac cct ggt tat acg tat ata 1152Lys Asp Ala Val Leu Leu Asp Ile Val Tyr Pro Gly Tyr Thr Tyr Ile 370 375380 ttt ttt gga atg cca gaa gtc gag ttt ttc atg gta aac caa ttg aat 1200Phe Phe Gly Met Pro Glu Val Glu Phe Phe Met Val Asn Gln Leu Asn 385 390395 400 aat acc aga aag acg tta aag tat aat cca gtt tcc aaa gat att ata1248 Asn Thr Arg Lys Thr Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile 405410 415 gcg agt aca aga gat tcg gaa tta gaa tta cct cca gaa act tca gat1296 Ala Ser Thr Arg Asp Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp 420425 430 caa cca aat tat gag tca tat agc cat aga tta tgt cat atc aca agt1344 Gln Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser 435440 445 att ccc gcg acg ggt aac act acc gga tta gta cct gta ttt tct tgg1392 Ile Pro Ala Thr Gly Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp 450455 460 aca cat cga agt gca gat tta aac aat aca ata tat tca gat aaa atc1440 Thr His Arg Ser Ala Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile 465470 475 480 act caa att ccg gcc gtt aaa tgt tgg gat aat tta ccg ttt gttcca 1488 Thr Gln Ile Pro Ala Val Lys Cys Trp Asp Asn Leu Pro Phe Val Pro485 490 495 gtg gta aaa gga cca gga cat aca gga ggg gat tta tta cag tataat 1536 Val Val Lys Gly Pro Gly His Thr Gly Gly Asp Leu Leu Gln Tyr Asn500 505 510 aga agt act ggt tct gta gga acc tta ttt cta gct cga tat ggccta 1584 Arg Ser Thr Gly Ser Val Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu515 520 525 gca tta gaa aaa gca ggg aaa tat cgt gta aga ctg aga tat gctact 1632 Ala Leu Glu Lys Ala Gly Lys Tyr Arg Val Arg Leu Arg Tyr Ala Thr530 535 540 gat gca gat att gta ttg cat gta aac gat gct cag att cag atgcca 1680 Asp Ala Asp Ile Val Leu His Val Asn Asp Ala Gln Ile Gln Met Pro545 550 555 560 aaa aca atg aac cca ggt gag gat ctg aca tct aaa act tttaaa gtt 1728 Lys Thr Met Asn Pro Gly Glu Asp Leu Thr Ser Lys Thr Phe LysVal 565 570 575 gca gat gct atc aca aca tta aat tta gca aca gat agt tcgcta gca 1776 Ala Asp Ala Ile Thr Thr Leu Asn Leu Ala Thr Asp Ser Ser LeuAla 580 585 590 ttg aaa cat aat tta ggt gaa gac cct aat tca aca tta tctggt ata 1824 Leu Lys His Asn Leu Gly Glu Asp Pro Asn Ser Thr Leu Ser GlyIle 595 600 605 gtt tac gtt gac cga atc gaa ttc atc cca gta gat taa 1863Val Tyr Val Asp Arg Ile Glu Phe Ile Pro Val Asp * 610 615 620 <210> SEQID NO 30 <211> LENGTH: 620 <212> TYPE: PRT <213> ORGANISM: Bacillusthuringiensis (mutated) <400> SEQUENCE: 30 Met Ser Ala Gly Asn Ala SerGlu Tyr Pro Gly Ser Pro Glu Val Leu 1 5 10 15 Val Ser Gly Gln Asp AlaAla Lys Ala Ala Ile Asp Ile Val Gly Lys 20 25 30 Leu Leu Ser Gly Leu GlyVal Pro Phe Val Gly Pro Ile Val Ser Leu 35 40 45 Tyr Thr Gln Leu Ile AspIle Leu Trp Pro Ser Gly Glu Lys Ser Gln 50 55 60 Trp Glu Ile Phe Met GluGln Val Glu Glu Leu Ile Asn Gln Lys Ile 65 70 75 80 Ala Glu Tyr Ala ArgAsn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly 85 90 95 Asn Asn Tyr Gln LeuTyr Leu Thr Ala Leu Glu Glu Trp Glu Glu Asn 100 105 110 Pro Asn Gly SerArg Asn Gly Ser Arg Ala Leu Arg Asp Val Arg Asn 115 120 125 Arg Phe GluIle Leu Asp Ser Leu Phe Thr Gln Tyr Met Pro Ser Phe 130 135 140 Arg ValThr Asn Phe Glu Val Pro Phe Leu Thr Val Tyr Ala Met Ala 145 150 155 160Ala Asn Leu His Leu Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu 165 170175 Glu Trp Gly Trp Ser Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln 180185 190 Met Lys Leu Thr Ala Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu195 200 205 Thr Gly Leu Ala Lys Leu Lys Gly Thr Ser Ala Lys Gln Trp ValAsp 210 215 220 Tyr Asn Gln Phe Arg Arg Glu Met Thr Leu Ala Val Leu AspVal Val 225 230 235 240 Ala Leu Phe Pro Asn Tyr Asp Thr Arg Thr Tyr ProMet Glu Thr Lys 245 250 255 Ala Gln Leu Thr Arg Glu Val Tyr Thr Asp ProLeu Gly Ala Val Asn 260 265 270 Val Ser Ser Ile Gly Ser Trp Tyr Asp LysAla Pro Ser Phe Gly Val 275 280 285 Ile Glu Ser Ser Val Ile Arg Pro ProHis Val Phe Asp Tyr Ile Thr 290 295 300 Gly Leu Thr Val Tyr Thr Gln SerArg Ser Ile Ser Ser Ala Arg Tyr 305 310 315 320 Ile Arg His Trp Ala GlyHis Gln Ile Ser Tyr His Arg Val Ser Arg 325 330 335 Gly Ser Asn Leu GlnGln Met Tyr Gly Thr Asn Gln Asn Leu His Ser 340 345 350 Thr Ser Thr PheAsp Phe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser 355 360 365 Lys Asp AlaVal Leu Leu Asp Ile Val Tyr Pro Gly Tyr Thr Tyr Ile 370 375 380 Phe PheGly Met Pro Glu Val Glu Phe Phe Met Val Asn Gln Leu Asn 385 390 395 400Asn Thr Arg Lys Thr Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile 405 410415 Ala Ser Thr Arg Asp Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp 420425 430 Gln Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser435 440 445 Ile Pro Ala Thr Gly Asn Thr Thr Gly Leu Val Pro Val Phe SerTrp 450 455 460 Thr His Arg Ser Ala Asp Leu Asn Asn Thr Ile Tyr Ser AspLys Ile 465 470 475 480 Thr Gln Ile Pro Ala Val Lys Cys Trp Asp Asn LeuPro Phe Val Pro 485 490 495 Val Val Lys Gly Pro Gly His Thr Gly Gly AspLeu Leu Gln Tyr Asn 500 505 510 Arg Ser Thr Gly Ser Val Gly Thr Leu PheLeu Ala Arg Tyr Gly Leu 515 520 525 Ala Leu Glu Lys Ala Gly Lys Tyr ArgVal Arg Leu Arg Tyr Ala Thr 530 535 540 Asp Ala Asp Ile Val Leu His ValAsn Asp Ala Gln Ile Gln Met Pro 545 550 555 560 Lys Thr Met Asn Pro GlyGlu Asp Leu Thr Ser Lys Thr Phe Lys Val 565 570 575 Ala Asp Ala Ile ThrThr Leu Asn Leu Ala Thr Asp Ser Ser Leu Ala 580 585 590 Leu Lys His AsnLeu Gly Glu Asp Pro Asn Ser Thr Leu Ser Gly Ile 595 600 605 Val Tyr ValAsp Arg Ile Glu Phe Ile Pro Val Asp 610 615 620 <210> SEQ ID NO 31 <211>LENGTH: 1863 <212> TYPE: DNA <213> ORGANISM: Bacillus thuringiensis(mutated) <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)...(1863) <221> NAME/KEY: misc_feature <222> LOCATION: (0)...(0)<223> OTHER INFORMATION: LKMS.N49PVD <400> SEQUENCE: 31 atg tct gcg ggaaat gct agt gaa tac cct ggt tca cct gaa gta ctt 48 Met Ser Ala Gly AsnAla Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu 1 5 10 15 gtt agc gga caagat gca gct aag gcc gca att gat ata gta ggt aaa 96 Val Ser Gly Gln AspAla Ala Lys Ala Ala Ile Asp Ile Val Gly Lys 20 25 30 tta cta tca ggt ttaggg gtc cca ttt gtt ggg ccg ata gtg agt ctt 144 Leu Leu Ser Gly Leu GlyVal Pro Phe Val Gly Pro Ile Val Ser Leu 35 40 45 tat act caa ctt att gatatt ctg tgg cct tca ggg gaa aag agt caa 192 Tyr Thr Gln Leu Ile Asp IleLeu Trp Pro Ser Gly Glu Lys Ser Gln 50 55 60 tgg gaa att ttt atg gaa caagta gaa gaa ctc att aat caa aaa ata 240 Trp Glu Ile Phe Met Glu Gln ValGlu Glu Leu Ile Asn Gln Lys Ile 65 70 75 80 gca gaa tat gca agg aat aaagcg ctt tcg gaa tta gaa gga tta ggt 288 Ala Glu Tyr Ala Arg Asn Lys AlaLeu Ser Glu Leu Glu Gly Leu Gly 85 90 95 aat aat tac caa tta tat cta actgcg ctt gaa gaa tgg gaa gaa aat 336 Asn Asn Tyr Gln Leu Tyr Leu Thr AlaLeu Glu Glu Trp Glu Glu Asn 100 105 110 cca tta aaa atg tct aat ggt tctaga gcc tta cga gat gtg cga aat 384 Pro Leu Lys Met Ser Asn Gly Ser ArgAla Leu Arg Asp Val Arg Asn 115 120 125 cga ttt gaa atc ctg gat agt ttattt acg caa tat atg cca tct ttt 432 Arg Phe Glu Ile Leu Asp Ser Leu PheThr Gln Tyr Met Pro Ser Phe 130 135 140 aga gtg aca aat ttt gaa gta ccattc ctt act gta tat gca atg gca 480 Arg Val Thr Asn Phe Glu Val Pro PheLeu Thr Val Tyr Ala Met Ala 145 150 155 160 gcc aac ctt cat tta ctg ttatta aag gac gcg tca att ttt gga gaa 528 Ala Asn Leu His Leu Leu Leu LeuLys Asp Ala Ser Ile Phe Gly Glu 165 170 175 gaa tgg gga tgg tca aca actact att aat aac tat tat gat cgt caa 576 Glu Trp Gly Trp Ser Thr Thr ThrIle Asn Asn Tyr Tyr Asp Arg Gln 180 185 190 atg aaa ctt act gca gaa tattct gat cac tgt gta aag tgg tat gaa 624 Met Lys Leu Thr Ala Glu Tyr SerAsp His Cys Val Lys Trp Tyr Glu 195 200 205 act ggt tta gca aaa tta aaaggc acg agc gct aaa caa tgg gtt gac 672 Thr Gly Leu Ala Lys Leu Lys GlyThr Ser Ala Lys Gln Trp Val Asp 210 215 220 tat aac caa ttc cgt aga gaaatg aca ctg gcg gtt tta gat gtt gtt 720 Tyr Asn Gln Phe Arg Arg Glu MetThr Leu Ala Val Leu Asp Val Val 225 230 235 240 gca tta ttc cca aat tatgac aca cgc acg tac cca atg gaa acg aaa 768 Ala Leu Phe Pro Asn Tyr AspThr Arg Thr Tyr Pro Met Glu Thr Lys 245 250 255 gca caa cta aca agg gaagta tat aca gat cca ctg ggc gcg gta aac 816 Ala Gln Leu Thr Arg Glu ValTyr Thr Asp Pro Leu Gly Ala Val Asn 260 265 270 gtg tct tca att ggt tcctgg tat gac aaa gca cct tct ttc gga gtg 864 Val Ser Ser Ile Gly Ser TrpTyr Asp Lys Ala Pro Ser Phe Gly Val 275 280 285 ata gaa tca tcc gtt attcga cca ccc cat gta ttt gat tat ata acg 912 Ile Glu Ser Ser Val Ile ArgPro Pro His Val Phe Asp Tyr Ile Thr 290 295 300 gga ctc aca gtg tat acacaa tca aga agc att tct tcc gct cgc tat 960 Gly Leu Thr Val Tyr Thr GlnSer Arg Ser Ile Ser Ser Ala Arg Tyr 305 310 315 320 ata aga cat tgg gctggt cat caa ata agc tac cat cgt gtc agt agg 1008 Ile Arg His Trp Ala GlyHis Gln Ile Ser Tyr His Arg Val Ser Arg 325 330 335 ggt agt aat ctt caacaa atg tat gga act aat caa aat cta cac agc 1056 Gly Ser Asn Leu Gln GlnMet Tyr Gly Thr Asn Gln Asn Leu His Ser 340 345 350 act agt acc ttt gatttt acg aat tat gat att tac aag act cta tca 1104 Thr Ser Thr Phe Asp PheThr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser 355 360 365 aag gat gca gta ctcctt gat att gtt tac cct ggt tat acg tat ata 1152 Lys Asp Ala Val Leu LeuAsp Ile Val Tyr Pro Gly Tyr Thr Tyr Ile 370 375 380 ttt ttt gga atg ccagaa gtc gag ttt ttc atg gta aac caa ttg aat 1200 Phe Phe Gly Met Pro GluVal Glu Phe Phe Met Val Asn Gln Leu Asn 385 390 395 400 aat acc aga aagacg tta aag tat aat cca gtt tcc aaa gat att ata 1248 Asn Thr Arg Lys ThrLeu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile 405 410 415 gcg agt aca agagat tcg gaa tta gaa tta cct cca gaa act tca gat 1296 Ala Ser Thr Arg AspSer Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp 420 425 430 caa cca aat tatgag tca tat agc cat aga tta tgt cat atc aca agt 1344 Gln Pro Asn Tyr GluSer Tyr Ser His Arg Leu Cys His Ile Thr Ser 435 440 445 att ccc gcg acgggt aac act acc gga tta gta cct gta ttt tct tgg 1392 Ile Pro Ala Thr GlyAsn Thr Thr Gly Leu Val Pro Val Phe Ser Trp 450 455 460 aca cat cga agtgca gat tta aac aat aca ata tat tca gat aaa atc 1440 Thr His Arg Ser AlaAsp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile 465 470 475 480 act caa attccg gcc gtt aaa tgt tgg gat aat tta ccg ttt gtt cca 1488 Thr Gln Ile ProAla Val Lys Cys Trp Asp Asn Leu Pro Phe Val Pro 485 490 495 gtg gta aaagga cca gga cat aca gga ggg gat tta tta cag tat aat 1536 Val Val Lys GlyPro Gly His Thr Gly Gly Asp Leu Leu Gln Tyr Asn 500 505 510 aga agt actggt tct gta gga acc tta ttt cta gct cga tat ggc cta 1584 Arg Ser Thr GlySer Val Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu 515 520 525 gca tta gaaaaa gca ggg aaa tat cgt gta aga ctg aga tat gct act 1632 Ala Leu Glu LysAla Gly Lys Tyr Arg Val Arg Leu Arg Tyr Ala Thr 530 535 540 gat gca gatatt gta ttg cat gta aac gat gct cag att cag atg cca 1680 Asp Ala Asp IleVal Leu His Val Asn Asp Ala Gln Ile Gln Met Pro 545 550 555 560 aaa acaatg aac cca ggt gag gat ctg aca tct aaa act ttt aaa gtt 1728 Lys Thr MetAsn Pro Gly Glu Asp Leu Thr Ser Lys Thr Phe Lys Val 565 570 575 gca gatgct atc aca aca tta aat tta gca aca gat agt tcg cta gca 1776 Ala Asp AlaIle Thr Thr Leu Asn Leu Ala Thr Asp Ser Ser Leu Ala 580 585 590 ttg aaacat aat tta ggt gaa gac cct aat tca aca tta tct ggt ata 1824 Leu Lys HisAsn Leu Gly Glu Asp Pro Asn Ser Thr Leu Ser Gly Ile 595 600 605 gtt tacgtt gac cga atc gaa ttc atc cca gta gat taa 1863 Val Tyr Val Asp Arg IleGlu Phe Ile Pro Val Asp * 610 615 620 <210> SEQ ID NO 32 <211> LENGTH:620 <212> TYPE: PRT <213> ORGANISM: Bacillus thuringiensis (mutated)<400> SEQUENCE: 32 Met Ser Ala Gly Asn Ala Ser Glu Tyr Pro Gly Ser ProGlu Val Leu 1 5 10 15 Val Ser Gly Gln Asp Ala Ala Lys Ala Ala Ile AspIle Val Gly Lys 20 25 30 Leu Leu Ser Gly Leu Gly Val Pro Phe Val Gly ProIle Val Ser Leu 35 40 45 Tyr Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser GlyGlu Lys Ser Gln 50 55 60 Trp Glu Ile Phe Met Glu Gln Val Glu Glu Leu IleAsn Gln Lys Ile 65 70 75 80 Ala Glu Tyr Ala Arg Asn Lys Ala Leu Ser GluLeu Glu Gly Leu Gly 85 90 95 Asn Asn Tyr Gln Leu Tyr Leu Thr Ala Leu GluGlu Trp Glu Glu Asn 100 105 110 Pro Leu Lys Met Ser Asn Gly Ser Arg AlaLeu Arg Asp Val Arg Asn 115 120 125 Arg Phe Glu Ile Leu Asp Ser Leu PheThr Gln Tyr Met Pro Ser Phe 130 135 140 Arg Val Thr Asn Phe Glu Val ProPhe Leu Thr Val Tyr Ala Met Ala 145 150 155 160 Ala Asn Leu His Leu LeuLeu Leu Lys Asp Ala Ser Ile Phe Gly Glu 165 170 175 Glu Trp Gly Trp SerThr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln 180 185 190 Met Lys Leu ThrAla Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu 195 200 205 Thr Gly LeuAla Lys Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp 210 215 220 Tyr AsnGln Phe Arg Arg Glu Met Thr Leu Ala Val Leu Asp Val Val 225 230 235 240Ala Leu Phe Pro Asn Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys 245 250255 Ala Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Gly Ala Val Asn 260265 270 Val Ser Ser Ile Gly Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val275 280 285 Ile Glu Ser Ser Val Ile Arg Pro Pro His Val Phe Asp Tyr IleThr 290 295 300 Gly Leu Thr Val Tyr Thr Gln Ser Arg Ser Ile Ser Ser AlaArg Tyr 305 310 315 320 Ile Arg His Trp Ala Gly His Gln Ile Ser Tyr HisArg Val Ser Arg 325 330 335 Gly Ser Asn Leu Gln Gln Met Tyr Gly Thr AsnGln Asn Leu His Ser 340 345 350 Thr Ser Thr Phe Asp Phe Thr Asn Tyr AspIle Tyr Lys Thr Leu Ser 355 360 365 Lys Asp Ala Val Leu Leu Asp Ile ValTyr Pro Gly Tyr Thr Tyr Ile 370 375 380 Phe Phe Gly Met Pro Glu Val GluPhe Phe Met Val Asn Gln Leu Asn 385 390 395 400 Asn Thr Arg Lys Thr LeuLys Tyr Asn Pro Val Ser Lys Asp Ile Ile 405 410 415 Ala Ser Thr Arg AspSer Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp 420 425 430 Gln Pro Asn TyrGlu Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser 435 440 445 Ile Pro AlaThr Gly Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp 450 455 460 Thr HisArg Ser Ala Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile 465 470 475 480Thr Gln Ile Pro Ala Val Lys Cys Trp Asp Asn Leu Pro Phe Val Pro 485 490495 Val Val Lys Gly Pro Gly His Thr Gly Gly Asp Leu Leu Gln Tyr Asn 500505 510 Arg Ser Thr Gly Ser Val Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu515 520 525 Ala Leu Glu Lys Ala Gly Lys Tyr Arg Val Arg Leu Arg Tyr AlaThr 530 535 540 Asp Ala Asp Ile Val Leu His Val Asn Asp Ala Gln Ile GlnMet Pro 545 550 555 560 Lys Thr Met Asn Pro Gly Glu Asp Leu Thr Ser LysThr Phe Lys Val 565 570 575 Ala Asp Ala Ile Thr Thr Leu Asn Leu Ala ThrAsp Ser Ser Leu Ala 580 585 590 Leu Lys His Asn Leu Gly Glu Asp Pro AsnSer Thr Leu Ser Gly Ile 595 600 605 Val Tyr Val Asp Arg Ile Glu Phe IlePro Val Asp 610 615 620 <210> SEQ ID NO 33 <211> LENGTH: 1854 <212>TYPE: DNA <213> ORGANISM: Bacillus thuringiensis (mutated) <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(1854) <221>NAME/KEY: misc_feature <222> LOCATION: (0)...(0) <223> OTHERINFORMATION: LKMS.R49PVD <400> SEQUENCE: 33 atg tct gcg gga aat gct agtgaa tac cct ggt tca cct gaa gta ctt 48 Met Ser Ala Gly Asn Ala Ser GluTyr Pro Gly Ser Pro Glu Val Leu 1 5 10 15 gtt agc gga caa gat gca gctaag gcc gca att gat ata gta ggt aaa 96 Val Ser Gly Gln Asp Ala Ala LysAla Ala Ile Asp Ile Val Gly Lys 20 25 30 tta cta tca ggt tta ggg gtc ccattt gtt ggg ccg ata gtg agt ctt 144 Leu Leu Ser Gly Leu Gly Val Pro PheVal Gly Pro Ile Val Ser Leu 35 40 45 tat act caa ctt att gat att ctg tggcct tca ggg gaa aag agt caa 192 Tyr Thr Gln Leu Ile Asp Ile Leu Trp ProSer Gly Glu Lys Ser Gln 50 55 60 tgg gaa att ttt atg gaa caa gta gaa gaactc att aat caa aaa ata 240 Trp Glu Ile Phe Met Glu Gln Val Glu Glu LeuIle Asn Gln Lys Ile 65 70 75 80 gca gaa tat gca agg aat aaa gcg ctt tcggaa tta gaa gga tta ggt 288 Ala Glu Tyr Ala Arg Asn Lys Ala Leu Ser GluLeu Glu Gly Leu Gly 85 90 95 aat aat tac caa tta tat cta act gcg ctt gaagaa tgg gaa gaa aat 336 Asn Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu GluTrp Glu Glu Asn 100 105 110 cca tta aaa atg tct aga gcc tta cga gat gtgcga aat cga ttt gaa 384 Pro Leu Lys Met Ser Arg Ala Leu Arg Asp Val ArgAsn Arg Phe Glu 115 120 125 atc ctg gat agt tta ttt acg caa tat atg ccatct ttt aga gtg aca 432 Ile Leu Asp Ser Leu Phe Thr Gln Tyr Met Pro SerPhe Arg Val Thr 130 135 140 aat ttt gaa gta cca ttc ctt act gta tat gcaatg gca gcc aac ctt 480 Asn Phe Glu Val Pro Phe Leu Thr Val Tyr Ala MetAla Ala Asn Leu 145 150 155 160 cat tta ctg tta tta aag gac gcg tca attttt gga gaa gaa tgg gga 528 His Leu Leu Leu Leu Lys Asp Ala Ser Ile PheGly Glu Glu Trp Gly 165 170 175 tgg tca aca act act att aat aac tat tatgat cgt caa atg aaa ctt 576 Trp Ser Thr Thr Thr Ile Asn Asn Tyr Tyr AspArg Gln Met Lys Leu 180 185 190 act gca gaa tat tct gat cac tgt gta aagtgg tat gaa act ggt tta 624 Thr Ala Glu Tyr Ser Asp His Cys Val Lys TrpTyr Glu Thr Gly Leu 195 200 205 gca aaa tta aaa ggc acg agc gct aaa caatgg gtt gac tat aac caa 672 Ala Lys Leu Lys Gly Thr Ser Ala Lys Gln TrpVal Asp Tyr Asn Gln 210 215 220 ttc cgt aga gaa atg aca ctg gcg gtt ttagat gtt gtt gca tta ttc 720 Phe Arg Arg Glu Met Thr Leu Ala Val Leu AspVal Val Ala Leu Phe 225 230 235 240 cca aat tat gac aca cgc acg tac ccaatg gaa acg aaa gca caa cta 768 Pro Asn Tyr Asp Thr Arg Thr Tyr Pro MetGlu Thr Lys Ala Gln Leu 245 250 255 aca agg gaa gta tat aca gat cca ctgggc gcg gta aac gtg tct tca 816 Thr Arg Glu Val Tyr Thr Asp Pro Leu GlyAla Val Asn Val Ser Ser 260 265 270 att ggt tcc tgg tat gac aaa gca ccttct ttc gga gtg ata gaa tca 864 Ile Gly Ser Trp Tyr Asp Lys Ala Pro SerPhe Gly Val Ile Glu Ser 275 280 285 tcc gtt att cga cca ccc cat gta tttgat tat ata acg gga ctc aca 912 Ser Val Ile Arg Pro Pro His Val Phe AspTyr Ile Thr Gly Leu Thr 290 295 300 gtg tat aca caa tca aga agc att tcttcc gct cgc tat ata aga cat 960 Val Tyr Thr Gln Ser Arg Ser Ile Ser SerAla Arg Tyr Ile Arg His 305 310 315 320 tgg gct ggt cat caa ata agc taccat cgt gtc agt agg ggt agt aat 1008 Trp Ala Gly His Gln Ile Ser Tyr HisArg Val Ser Arg Gly Ser Asn 325 330 335 ctt caa caa atg tat gga act aatcaa aat cta cac agc act agt acc 1056 Leu Gln Gln Met Tyr Gly Thr Asn GlnAsn Leu His Ser Thr Ser Thr 340 345 350 ttt gat ttt acg aat tat gat atttac aag act cta tca aag gat gca 1104 Phe Asp Phe Thr Asn Tyr Asp Ile TyrLys Thr Leu Ser Lys Asp Ala 355 360 365 gta ctc ctt gat att gtt tac cctggt tat acg tat ata ttt ttt gga 1152 Val Leu Leu Asp Ile Val Tyr Pro GlyTyr Thr Tyr Ile Phe Phe Gly 370 375 380 atg cca gaa gtc gag ttt ttc atggta aac caa ttg aat aat acc aga 1200 Met Pro Glu Val Glu Phe Phe Met ValAsn Gln Leu Asn Asn Thr Arg 385 390 395 400 aag acg tta aag tat aat ccagtt tcc aaa gat att ata gcg agt aca 1248 Lys Thr Leu Lys Tyr Asn Pro ValSer Lys Asp Ile Ile Ala Ser Thr 405 410 415 aga gat tcg gaa tta gaa ttacct cca gaa act tca gat caa cca aat 1296 Arg Asp Ser Glu Leu Glu Leu ProPro Glu Thr Ser Asp Gln Pro Asn 420 425 430 tat gag tca tat agc cat agatta tgt cat atc aca agt att ccc gcg 1344 Tyr Glu Ser Tyr Ser His Arg LeuCys His Ile Thr Ser Ile Pro Ala 435 440 445 acg ggt aac act acc gga ttagta cct gta ttt tct tgg aca cat cga 1392 Thr Gly Asn Thr Thr Gly Leu ValPro Val Phe Ser Trp Thr His Arg 450 455 460 agt gca gat tta aac aat acaata tat tca gat aaa atc act caa att 1440 Ser Ala Asp Leu Asn Asn Thr IleTyr Ser Asp Lys Ile Thr Gln Ile 465 470 475 480 ccg gcc gtt aaa tgt tgggat aat tta ccg ttt gtt cca gtg gta aaa 1488 Pro Ala Val Lys Cys Trp AspAsn Leu Pro Phe Val Pro Val Val Lys 485 490 495 gga cca gga cat aca ggaggg gat tta tta cag tat aat aga agt act 1536 Gly Pro Gly His Thr Gly GlyAsp Leu Leu Gln Tyr Asn Arg Ser Thr 500 505 510 ggt tct gta gga acc ttattt cta gct cga tat ggc cta gca tta gaa 1584 Gly Ser Val Gly Thr Leu PheLeu Ala Arg Tyr Gly Leu Ala Leu Glu 515 520 525 aaa gca ggg aaa tat cgtgta aga ctg aga tat gct act gat gca gat 1632 Lys Ala Gly Lys Tyr Arg ValArg Leu Arg Tyr Ala Thr Asp Ala Asp 530 535 540 att gta ttg cat gta aacgat gct cag att cag atg cca aaa aca atg 1680 Ile Val Leu His Val Asn AspAla Gln Ile Gln Met Pro Lys Thr Met 545 550 555 560 aac cca ggt gag gatctg aca tct aaa act ttt aaa gtt gca gat gct 1728 Asn Pro Gly Glu Asp LeuThr Ser Lys Thr Phe Lys Val Ala Asp Ala 565 570 575 atc aca aca tta aattta gca aca gat agt tcg cta gca ttg aaa cat 1776 Ile Thr Thr Leu Asn LeuAla Thr Asp Ser Ser Leu Ala Leu Lys His 580 585 590 aat tta ggt gaa gaccct aat tca aca tta tct ggt ata gtt tac gtt 1824 Asn Leu Gly Glu Asp ProAsn Ser Thr Leu Ser Gly Ile Val Tyr Val 595 600 605 gac cga atc gaa ttcatc cca gta gat taa 1854 Asp Arg Ile Glu Phe Ile Pro Val Asp * 610 615<210> SEQ ID NO 34 <211> LENGTH: 617 <212> TYPE: PRT <213> ORGANISM:Bacillus thuringiensis (mutated) <400> SEQUENCE: 34 Met Ser Ala Gly AsnAla Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu 1 5 10 15 Val Ser Gly GlnAsp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly Lys 20 25 30 Leu Leu Ser GlyLeu Gly Val Pro Phe Val Gly Pro Ile Val Ser Leu 35 40 45 Tyr Thr Gln LeuIle Asp Ile Leu Trp Pro Ser Gly Glu Lys Ser Gln 50 55 60 Trp Glu Ile PheMet Glu Gln Val Glu Glu Leu Ile Asn Gln Lys Ile 65 70 75 80 Ala Glu TyrAla Arg Asn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly 85 90 95 Asn Asn TyrGln Leu Tyr Leu Thr Ala Leu Glu Glu Trp Glu Glu Asn 100 105 110 Pro LeuLys Met Ser Arg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu 115 120 125 IleLeu Asp Ser Leu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr 130 135 140Asn Phe Glu Val Pro Phe Leu Thr Val Tyr Ala Met Ala Ala Asn Leu 145 150155 160 His Leu Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly165 170 175 Trp Ser Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met LysLeu 180 185 190 Thr Ala Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu ThrGly Leu 195 200 205 Ala Lys Leu Lys Gly Thr Ser Ala Lys Gln Trp Val AspTyr Asn Gln 210 215 220 Phe Arg Arg Glu Met Thr Leu Ala Val Leu Asp ValVal Ala Leu Phe 225 230 235 240 Pro Asn Tyr Asp Thr Arg Thr Tyr Pro MetGlu Thr Lys Ala Gln Leu 245 250 255 Thr Arg Glu Val Tyr Thr Asp Pro LeuGly Ala Val Asn Val Ser Ser 260 265 270 Ile Gly Ser Trp Tyr Asp Lys AlaPro Ser Phe Gly Val Ile Glu Ser 275 280 285 Ser Val Ile Arg Pro Pro HisVal Phe Asp Tyr Ile Thr Gly Leu Thr 290 295 300 Val Tyr Thr Gln Ser ArgSer Ile Ser Ser Ala Arg Tyr Ile Arg His 305 310 315 320 Trp Ala Gly HisGln Ile Ser Tyr His Arg Val Ser Arg Gly Ser Asn 325 330 335 Leu Gln GlnMet Tyr Gly Thr Asn Gln Asn Leu His Ser Thr Ser Thr 340 345 350 Phe AspPhe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala 355 360 365 ValLeu Leu Asp Ile Val Tyr Pro Gly Tyr Thr Tyr Ile Phe Phe Gly 370 375 380Met Pro Glu Val Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg 385 390395 400 Lys Thr Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Ser Thr405 410 415 Arg Asp Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln ProAsn 420 425 430 Tyr Glu Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser IlePro Ala 435 440 445 Thr Gly Asn Thr Thr Gly Leu Val Pro Val Phe Ser TrpThr His Arg 450 455 460 Ser Ala Asp Leu Asn Asn Thr Ile Tyr Ser Asp LysIle Thr Gln Ile 465 470 475 480 Pro Ala Val Lys Cys Trp Asp Asn Leu ProPhe Val Pro Val Val Lys 485 490 495 Gly Pro Gly His Thr Gly Gly Asp LeuLeu Gln Tyr Asn Arg Ser Thr 500 505 510 Gly Ser Val Gly Thr Leu Phe LeuAla Arg Tyr Gly Leu Ala Leu Glu 515 520 525 Lys Ala Gly Lys Tyr Arg ValArg Leu Arg Tyr Ala Thr Asp Ala Asp 530 535 540 Ile Val Leu His Val AsnAsp Ala Gln Ile Gln Met Pro Lys Thr Met 545 550 555 560 Asn Pro Gly GluAsp Leu Thr Ser Lys Thr Phe Lys Val Ala Asp Ala 565 570 575 Ile Thr ThrLeu Asn Leu Ala Thr Asp Ser Ser Leu Ala Leu Lys His 580 585 590 Asn LeuGly Glu Asp Pro Asn Ser Thr Leu Ser Gly Ile Val Tyr Val 595 600 605 AspArg Ile Glu Phe Ile Pro Val Asp 610 615 <210> SEQ ID NO 35 <211> LENGTH:22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: 5′ forward primer <400> SEQUENCE: 35 atgagtccaaataatcaaaa tg 22 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: 5′ reverse primer <400> SEQUENCE: 36 ccgcttctaa atcttgttcc20 <210> SEQ ID NO 37 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 3′ forwardprimer <400> SEQUENCE: 37 ggaacaagat ttagagg 17 <210> SEQ ID NO 38 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: 3′ reverse primer <400> SEQUENCE: 38ctcatcgtct acaatcaatt catc 24 <210> SEQ ID NO 39 <211> LENGTH: 2022<212> TYPE: DNA <213> ORGANISM: Bacillus thuringiensis (mutated) <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(2022) <221>NAME/KEY: misc_feature <222> LOCATION: (0)...(0) <223> OTHERINFORMATION: LRNS.N1218-1 <400> SEQUENCE: 39 atg agt cca aat aat caa aatgaa tat gaa att ata gat gcg aca cct 48 Met Ser Pro Asn Asn Gln Asn GluTyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 tct act tct gta tcc aat gattct aac aga tac cct ttt gcg aat gag 96 Ser Thr Ser Val Ser Asn Asp SerAsn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 cca aca aat gcg cta caa aat atggat tat aaa gat tat tta aaa atg 144 Pro Thr Asn Ala Leu Gln Asn Met AspTyr Lys Asp Tyr Leu Lys Met 35 40 45 tct gcg gga aat gct agt gaa tac cctggt tca cct gaa gta ctt gtt 192 Ser Ala Gly Asn Ala Ser Glu Tyr Pro GlySer Pro Glu Val Leu Val 50 55 60 agc gga caa gat gca gct aag gcc gca attgat ata gta ggt aaa tta 240 Ser Gly Gln Asp Ala Ala Lys Ala Ala Ile AspIle Val Gly Lys Leu 65 70 75 80 cta tca ggt tta ggg gtc cca ttt gtt gggccg ata gtg agt ctt tat 288 Leu Ser Gly Leu Gly Val Pro Phe Val Gly ProIle Val Ser Leu Tyr 85 90 95 act caa ctt att gat att ctg tgg cct tca ggggaa aag agt caa tgg 336 Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly GluLys Ser Gln Trp 100 105 110 gaa att ttt atg gaa caa gta gaa gaa ctc attaat caa aaa ata gca 384 Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile AsnGln Lys Ile Ala 115 120 125 gaa tat gca agg aat aaa gcg ctt tcg gaa ttagaa gga tta ggt aat 432 Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu GluGly Leu Gly Asn 130 135 140 aat tac caa tta tat cta act gcg ctt gaa gaatgg gaa gaa aat cca 480 Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu TrpGlu Glu Asn Pro 145 150 155 160 tta aga atg tct aat ggt tca aga gcc ttacga gat gtg cga aat cga 528 Leu Arg Met Ser Asn Gly Ser Arg Ala Leu ArgAsp Val Arg Asn Arg 165 170 175 ttt gaa atc ctg gat agt tta ttt acg caatat atg cca tct ttt aga 576 Phe Glu Ile Leu Asp Ser Leu Phe Thr Gln TyrMet Pro Ser Phe Arg 180 185 190 gtg aca aat ttt gaa gta cca ttc ctt actgta tat gca atg gca gcc 624 Val Thr Asn Phe Glu Val Pro Phe Leu Thr ValTyr Ala Met Ala Ala 195 200 205 aac ctt cat tta ctg tta tta aag gac gcgtca att ttt gga gaa gaa 672 Asn Leu His Leu Leu Leu Leu Lys Asp Ala SerIle Phe Gly Glu Glu 210 215 220 tgg gga tgg tca aca act act att aat aactat tat gat cgt caa atg 720 Trp Gly Trp Ser Thr Thr Thr Ile Asn Asn TyrTyr Asp Arg Gln Met 225 230 235 240 aaa ctt act gca gaa tat tct gat cactgt gta aag tgg tat gaa act 768 Lys Leu Thr Ala Glu Tyr Ser Asp His CysVal Lys Trp Tyr Glu Thr 245 250 255 ggt tta gca aaa tta aaa ggc acg agcgct aaa caa tgg gtt gac tat 816 Gly Leu Ala Lys Leu Lys Gly Thr Ser AlaLys Gln Trp Val Asp Tyr 260 265 270 aac caa ttc cgt aga gaa atg aca ctggcg gtt tta gat gtt gtt gca 864 Asn Gln Phe Arg Arg Glu Met Thr Leu AlaVal Leu Asp Val Val Ala 275 280 285 tta ttc cca aat tat gac aca cgc acgtac cca atg gaa acg aaa gca 912 Leu Phe Pro Asn Tyr Asp Thr Arg Thr TyrPro Met Glu Thr Lys Ala 290 295 300 caa cta aca agg gaa gta tat aca gatcca ctg ggc gcg gta aac gtg 960 Gln Leu Thr Arg Glu Val Tyr Thr Asp ProLeu Gly Ala Val Asn Val 305 310 315 320 tct tca att ggt tcc tgg tat gacaaa gca cct tct ttc gga gtg ata 1008 Ser Ser Ile Gly Ser Trp Tyr Asp LysAla Pro Ser Phe Gly Val Ile 325 330 335 gaa tca tcc gtt att cga cca ccccat gta ttt gat tat ata acg gga 1056 Glu Ser Ser Val Ile Arg Pro Pro HisVal Phe Asp Tyr Ile Thr Gly 340 345 350 ctc aca gtg tat aca caa tca agaagc att tct tcc gct cgc tat ata 1104 Leu Thr Val Tyr Thr Gln Ser Arg SerIle Ser Ser Ala Arg Tyr Ile 355 360 365 aga cat tgg gct ggt cat caa ataagc tac cat cgt gtc agt agg ggt 1152 Arg His Trp Ala Gly His Gln Ile SerTyr His Arg Val Ser Arg Gly 370 375 380 agt aat ctt caa caa atg tat ggaact aat caa aat cta cac agc act 1200 Ser Asn Leu Gln Gln Met Tyr Gly ThrAsn Gln Asn Leu His Ser Thr 385 390 395 400 agt acc ttt gat ttt acg aattat gat att tac aag act cta tca aag 1248 Ser Thr Phe Asp Phe Thr Asn TyrAsp Ile Tyr Lys Thr Leu Ser Lys 405 410 415 gat gca gta ctc ctt gat attgtt tac cct ggt tat acg tat ata ttt 1296 Asp Ala Val Leu Leu Asp Ile ValTyr Pro Gly Tyr Thr Tyr Ile Phe 420 425 430 ttt gga atg cca gaa gtc gagttt ttc atg gta aac caa ttg aat aat 1344 Phe Gly Met Pro Glu Val Glu PhePhe Met Val Asn Gln Leu Asn Asn 435 440 445 acc aga aag acg tta aag tataat cca gtt tcc aaa gat att ata gcg 1392 Thr Arg Lys Thr Leu Lys Tyr AsnPro Val Ser Lys Asp Ile Ile Ala 450 455 460 agt aca aga gat tcg gaa ttagaa tta cct cca gaa act tca gat caa 1440 Ser Thr Arg Asp Ser Glu Leu GluLeu Pro Pro Glu Thr Ser Asp Gln 465 470 475 480 cca aat tat gag tca tatagc cat aga tta tgt cat atc aca agt att 1488 Pro Asn Tyr Glu Ser Tyr SerHis Arg Leu Cys His Ile Thr Ser Ile 485 490 495 ccc gcg acg ggt aac actacc gga tta gta cct gta ttt tct tgg aca 1536 Pro Ala Thr Gly Asn Thr ThrGly Leu Val Pro Val Phe Ser Trp Thr 500 505 510 cat cga agt gca gat ttaaac aat aca ata tat tca gat aaa atc act 1584 His Arg Ser Ala Asp Leu AsnAsn Thr Ile Tyr Ser Asp Lys Ile Thr 515 520 525 caa att ccg gcc gtt aaatgt tgg gat aat tta ccg ttt gtt cca gtg 1632 Gln Ile Pro Ala Val Lys CysTrp Asp Asn Leu Pro Phe Val Pro Val 530 535 540 gta aaa gga cca gga cataca gga ggg gat tta tta cag tat aat aga 1680 Val Lys Gly Pro Gly His ThrGly Gly Asp Leu Leu Gln Tyr Asn Arg 545 550 555 560 agt act ggt tct gtagga acc tta ttt cta gct cga tat ggc cta gca 1728 Ser Thr Gly Ser Val GlyThr Leu Phe Leu Ala Arg Tyr Gly Leu Ala 565 570 575 tta gaa aaa gca gggaaa tat cgt gta aga ctg aga tat gct act gat 1776 Leu Glu Lys Ala Gly LysTyr Arg Val Arg Leu Arg Tyr Ala Thr Asp 580 585 590 gca gat att gta ttgcat gta aac gat gct cag att cag atg cca aaa 1824 Ala Asp Ile Val Leu HisVal Asn Asp Ala Gln Ile Gln Met Pro Lys 595 600 605 aca atg aac cca ggtgag gat ctg aca tct aaa act ttt aaa gtt gca 1872 Thr Met Asn Pro Gly GluAsp Leu Thr Ser Lys Thr Phe Lys Val Ala 610 615 620 gat gct atc aca acatta aat tta gca aca gat agt tcg cta gca ttg 1920 Asp Ala Ile Thr Thr LeuAsn Leu Ala Thr Asp Ser Ser Leu Ala Leu 625 630 635 640 aaa cat aat ttaggt gaa gac cct aat tca aca tta tct ggt ata gtt 1968 Lys His Asn Leu GlyGlu Asp Pro Asn Ser Thr Leu Ser Gly Ile Val 645 650 655 tac gtt gac cgaatc gaa ttc atc cca gta gat gag aca tat gaa gcg 2016 Tyr Val Asp Arg IleGlu Phe Ile Pro Val Asp Glu Thr Tyr Glu Ala 660 665 670 gaa taa 2022Glu * <210> SEQ ID NO 40 <211> LENGTH: 673 <212> TYPE: PRT <213>ORGANISM: Bacillus thuringiensis (mutated) <400> SEQUENCE: 40 Met SerPro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 SerThr Ser Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 ProThr Asn Ala Leu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45 SerAla Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 50 55 60 SerGly Gln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly Lys Leu 65 70 75 80Leu Ser Gly Leu Gly Val Pro Phe Val Gly Pro Ile Val Ser Leu Tyr 85 90 95Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly Glu Lys Ser Gln Trp 100 105110 Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile Asn Gln Lys Ile Ala 115120 125 Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly Asn130 135 140 Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu Trp Glu Glu AsnPro 145 150 155 160 Leu Arg Met Ser Asn Gly Ser Arg Ala Leu Arg Asp ValArg Asn Arg 165 170 175 Phe Glu Ile Leu Asp Ser Leu Phe Thr Gln Tyr MetPro Ser Phe Arg 180 185 190 Val Thr Asn Phe Glu Val Pro Phe Leu Thr ValTyr Ala Met Ala Ala 195 200 205 Asn Leu His Leu Leu Leu Leu Lys Asp AlaSer Ile Phe Gly Glu Glu 210 215 220 Trp Gly Trp Ser Thr Thr Thr Ile AsnAsn Tyr Tyr Asp Arg Gln Met 225 230 235 240 Lys Leu Thr Ala Glu Tyr SerAsp His Cys Val Lys Trp Tyr Glu Thr 245 250 255 Gly Leu Ala Lys Leu LysGly Thr Ser Ala Lys Gln Trp Val Asp Tyr 260 265 270 Asn Gln Phe Arg ArgGlu Met Thr Leu Ala Val Leu Asp Val Val Ala 275 280 285 Leu Phe Pro AsnTyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys Ala 290 295 300 Gln Leu ThrArg Glu Val Tyr Thr Asp Pro Leu Gly Ala Val Asn Val 305 310 315 320 SerSer Ile Gly Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val Ile 325 330 335Glu Ser Ser Val Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr Gly 340 345350 Leu Thr Val Tyr Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr Ile 355360 365 Arg His Trp Ala Gly His Gln Ile Ser Tyr His Arg Val Ser Arg Gly370 375 380 Ser Asn Leu Gln Gln Met Tyr Gly Thr Asn Gln Asn Leu His SerThr 385 390 395 400 Ser Thr Phe Asp Phe Thr Asn Tyr Asp Ile Tyr Lys ThrLeu Ser Lys 405 410 415 Asp Ala Val Leu Leu Asp Ile Val Tyr Pro Gly TyrThr Tyr Ile Phe 420 425 430 Phe Gly Met Pro Glu Val Glu Phe Phe Met ValAsn Gln Leu Asn Asn 435 440 445 Thr Arg Lys Thr Leu Lys Tyr Asn Pro ValSer Lys Asp Ile Ile Ala 450 455 460 Ser Thr Arg Asp Ser Glu Leu Glu LeuPro Pro Glu Thr Ser Asp Gln 465 470 475 480 Pro Asn Tyr Glu Ser Tyr SerHis Arg Leu Cys His Ile Thr Ser Ile 485 490 495 Pro Ala Thr Gly Asn ThrThr Gly Leu Val Pro Val Phe Ser Trp Thr 500 505 510 His Arg Ser Ala AspLeu Asn Asn Thr Ile Tyr Ser Asp Lys Ile Thr 515 520 525 Gln Ile Pro AlaVal Lys Cys Trp Asp Asn Leu Pro Phe Val Pro Val 530 535 540 Val Lys GlyPro Gly His Thr Gly Gly Asp Leu Leu Gln Tyr Asn Arg 545 550 555 560 SerThr Gly Ser Val Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu Ala 565 570 575Leu Glu Lys Ala Gly Lys Tyr Arg Val Arg Leu Arg Tyr Ala Thr Asp 580 585590 Ala Asp Ile Val Leu His Val Asn Asp Ala Gln Ile Gln Met Pro Lys 595600 605 Thr Met Asn Pro Gly Glu Asp Leu Thr Ser Lys Thr Phe Lys Val Ala610 615 620 Asp Ala Ile Thr Thr Leu Asn Leu Ala Thr Asp Ser Ser Leu AlaLeu 625 630 635 640 Lys His Asn Leu Gly Glu Asp Pro Asn Ser Thr Leu SerGly Ile Val 645 650 655 Tyr Val Asp Arg Ile Glu Phe Ile Pro Val Asp GluThr Tyr Glu Ala 660 665 670 Glu <210> SEQ ID NO 41 <211> LENGTH: 1863<212> TYPE: DNA <213> ORGANISM: Bacillus thuringiensis (mutated) <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(1863) <221>NAME/KEY: misc_feature <222> LOCATION: (0)...(0) <223> OTHERINFORMATION: LRMS.N49PVD <400> SEQUENCE: 41 atg tct gcg gga aat gct agtgaa tac cct ggt tca cct gaa gta ctt 48 Met Ser Ala Gly Asn Ala Ser GluTyr Pro Gly Ser Pro Glu Val Leu 1 5 10 15 gtt agc gga caa gat gca gctaag gcc gca att gat ata gta ggt aaa 96 Val Ser Gly Gln Asp Ala Ala LysAla Ala Ile Asp Ile Val Gly Lys 20 25 30 tta cta tca ggt tta ggg gtc ccattt gtt ggg ccg ata gtg agt ctt 144 Leu Leu Ser Gly Leu Gly Val Pro PheVal Gly Pro Ile Val Ser Leu 35 40 45 tat act caa ctt att gat att ctg tggcct tca ggg gaa aag agt caa 192 Tyr Thr Gln Leu Ile Asp Ile Leu Trp ProSer Gly Glu Lys Ser Gln 50 55 60 tgg gaa att ttt atg gaa caa gta gaa gaactc att aat caa aaa ata 240 Trp Glu Ile Phe Met Glu Gln Val Glu Glu LeuIle Asn Gln Lys Ile 65 70 75 80 gca gaa tat gca agg aat aaa gcg ctt tcggaa tta gaa gga tta ggt 288 Ala Glu Tyr Ala Arg Asn Lys Ala Leu Ser GluLeu Glu Gly Leu Gly 85 90 95 aat aat tac caa tta tat cta act gcg ctt gaagaa tgg gaa gaa aat 336 Asn Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu GluTrp Glu Glu Asn 100 105 110 cca tta aga atg tct aat ggt tcc cgg gcc ttacga gat gtg cga aat 384 Pro Leu Arg Met Ser Asn Gly Ser Arg Ala Leu ArgAsp Val Arg Asn 115 120 125 cga ttt gaa atc ctg gat agt tta ttt acg caatat atg cca tct ttt 432 Arg Phe Glu Ile Leu Asp Ser Leu Phe Thr Gln TyrMet Pro Ser Phe 130 135 140 aga gtg aca aat ttt gaa gta cca ttc ctt actgta tat gca atg gca 480 Arg Val Thr Asn Phe Glu Val Pro Phe Leu Thr ValTyr Ala Met Ala 145 150 155 160 gcc aac ctt cat tta ctg tta tta aag gacgcg tca att ttt gga gaa 528 Ala Asn Leu His Leu Leu Leu Leu Lys Asp AlaSer Ile Phe Gly Glu 165 170 175 gaa tgg gga tgg tca aca act act att aataac tat tat gat cgt caa 576 Glu Trp Gly Trp Ser Thr Thr Thr Ile Asn AsnTyr Tyr Asp Arg Gln 180 185 190 atg aaa ctt act gca gaa tat tct gat cactgt gta aag tgg tat gaa 624 Met Lys Leu Thr Ala Glu Tyr Ser Asp His CysVal Lys Trp Tyr Glu 195 200 205 act ggt tta gca aaa tta aaa ggc acg agcgct aaa caa tgg gtt gac 672 Thr Gly Leu Ala Lys Leu Lys Gly Thr Ser AlaLys Gln Trp Val Asp 210 215 220 tat aac caa ttc cgt aga gaa atg aca ctggcg gtt tta gat gtt gtt 720 Tyr Asn Gln Phe Arg Arg Glu Met Thr Leu AlaVal Leu Asp Val Val 225 230 235 240 gca tta ttc cca aat tat gac aca cgcacg tac cca atg gaa acg aaa 768 Ala Leu Phe Pro Asn Tyr Asp Thr Arg ThrTyr Pro Met Glu Thr Lys 245 250 255 gca caa cta aca agg gaa gta tat acagat cca ctg ggc gcg gta aac 816 Ala Gln Leu Thr Arg Glu Val Tyr Thr AspPro Leu Gly Ala Val Asn 260 265 270 gtg tct tca att ggt tcc tgg tat gacaaa gca cct tct ttc gga gtg 864 Val Ser Ser Ile Gly Ser Trp Tyr Asp LysAla Pro Ser Phe Gly Val 275 280 285 ata gaa tca tcc gtt att cga cca ccccat gta ttt gat tat ata acg 912 Ile Glu Ser Ser Val Ile Arg Pro Pro HisVal Phe Asp Tyr Ile Thr 290 295 300 gga ctc aca gtg tat aca caa tca agaagc att tct tcc gct cgc tat 960 Gly Leu Thr Val Tyr Thr Gln Ser Arg SerIle Ser Ser Ala Arg Tyr 305 310 315 320 ata aga cat tgg gct ggt cat caaata agc tac cat cgt gtc agt agg 1008 Ile Arg His Trp Ala Gly His Gln IleSer Tyr His Arg Val Ser Arg 325 330 335 ggt agt aat ctt caa caa atg tatgga act aat caa aat cta cac agc 1056 Gly Ser Asn Leu Gln Gln Met Tyr GlyThr Asn Gln Asn Leu His Ser 340 345 350 act agt acc ttt gat ttt acg aattat gat att tac aag act cta tca 1104 Thr Ser Thr Phe Asp Phe Thr Asn TyrAsp Ile Tyr Lys Thr Leu Ser 355 360 365 aag gat gca gta ctc ctt gat attgtt tac cct ggt tat acg tat ata 1152 Lys Asp Ala Val Leu Leu Asp Ile ValTyr Pro Gly Tyr Thr Tyr Ile 370 375 380 ttt ttt gga atg cca gaa gtc gagttt ttc atg gta aac caa ttg aat 1200 Phe Phe Gly Met Pro Glu Val Glu PhePhe Met Val Asn Gln Leu Asn 385 390 395 400 aat acc aga aag acg tta aagtat aat cca gtt tcc aaa gat att ata 1248 Asn Thr Arg Lys Thr Leu Lys TyrAsn Pro Val Ser Lys Asp Ile Ile 405 410 415 gcg agt aca aga gat tcg gaatta gaa tta cct cca gaa act tca gat 1296 Ala Ser Thr Arg Asp Ser Glu LeuGlu Leu Pro Pro Glu Thr Ser Asp 420 425 430 caa cca aat tat gag tca tatagc cat aga tta tgt cat atc aca agt 1344 Gln Pro Asn Tyr Glu Ser Tyr SerHis Arg Leu Cys His Ile Thr Ser 435 440 445 att ccc gcg acg ggt aac actacc gga tta gta cct gta ttt tct tgg 1392 Ile Pro Ala Thr Gly Asn Thr ThrGly Leu Val Pro Val Phe Ser Trp 450 455 460 aca cat cga agt gca gat ttaaac aat aca ata tat tca gat aaa atc 1440 Thr His Arg Ser Ala Asp Leu AsnAsn Thr Ile Tyr Ser Asp Lys Ile 465 470 475 480 act caa att ccg gcc gttaaa tgt tgg gat aat tta ccg ttt gtt cca 1488 Thr Gln Ile Pro Ala Val LysCys Trp Asp Asn Leu Pro Phe Val Pro 485 490 495 gtg gta aaa gga cca ggacat aca gga ggg gat tta tta cag tat aat 1536 Val Val Lys Gly Pro Gly HisThr Gly Gly Asp Leu Leu Gln Tyr Asn 500 505 510 aga agt act ggt tct gtagga acc tta ttt cta gct cga tat ggc cta 1584 Arg Ser Thr Gly Ser Val GlyThr Leu Phe Leu Ala Arg Tyr Gly Leu 515 520 525 gca tta gaa aaa gca gggaaa tat cgt gta aga ctg aga tat gct act 1632 Ala Leu Glu Lys Ala Gly LysTyr Arg Val Arg Leu Arg Tyr Ala Thr 530 535 540 gat gca gat att gta ttgcat gta aac gat gct cag att cag atg cca 1680 Asp Ala Asp Ile Val Leu HisVal Asn Asp Ala Gln Ile Gln Met Pro 545 550 555 560 aaa aca atg aac ccaggt gag gat ctg aca tct aaa act ttt aaa gtt 1728 Lys Thr Met Asn Pro GlyGlu Asp Leu Thr Ser Lys Thr Phe Lys Val 565 570 575 gca gat gct atc acaaca tta aat tta gca aca gat agt tcg cta gca 1776 Ala Asp Ala Ile Thr ThrLeu Asn Leu Ala Thr Asp Ser Ser Leu Ala 580 585 590 ttg aaa cat aat ttaggt gaa gac cct aat tca aca tta tct ggt ata 1824 Leu Lys His Asn Leu GlyGlu Asp Pro Asn Ser Thr Leu Ser Gly Ile 595 600 605 gtt tac gtt gac cgaatc gaa ttc atc cca gta gat taa 1863 Val Tyr Val Asp Arg Ile Glu Phe IlePro Val Asp * 610 615 620 <210> SEQ ID NO 42 <211> LENGTH: 620 <212>TYPE: PRT <213> ORGANISM: Bacillus thuringiensis (mutated) <400>SEQUENCE: 42 Met Ser Ala Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu ValLeu 1 5 10 15 Val Ser Gly Gln Asp Ala Ala Lys Ala Ala Ile Asp Ile ValGly Lys 20 25 30 Leu Leu Ser Gly Leu Gly Val Pro Phe Val Gly Pro Ile ValSer Leu 35 40 45 Tyr Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly Glu LysSer Gln 50 55 60 Trp Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile Asn GlnLys Ile 65 70 75 80 Ala Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu GluGly Leu Gly 85 90 95 Asn Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu TrpGlu Glu Asn 100 105 110 Pro Leu Arg Met Ser Asn Gly Ser Arg Ala Leu ArgAsp Val Arg Asn 115 120 125 Arg Phe Glu Ile Leu Asp Ser Leu Phe Thr GlnTyr Met Pro Ser Phe 130 135 140 Arg Val Thr Asn Phe Glu Val Pro Phe LeuThr Val Tyr Ala Met Ala 145 150 155 160 Ala Asn Leu His Leu Leu Leu LeuLys Asp Ala Ser Ile Phe Gly Glu 165 170 175 Glu Trp Gly Trp Ser Thr ThrThr Ile Asn Asn Tyr Tyr Asp Arg Gln 180 185 190 Met Lys Leu Thr Ala GluTyr Ser Asp His Cys Val Lys Trp Tyr Glu 195 200 205 Thr Gly Leu Ala LysLeu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp 210 215 220 Tyr Asn Gln PheArg Arg Glu Met Thr Leu Ala Val Leu Asp Val Val 225 230 235 240 Ala LeuPhe Pro Asn Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys 245 250 255 AlaGln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Gly Ala Val Asn 260 265 270Val Ser Ser Ile Gly Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val 275 280285 Ile Glu Ser Ser Val Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr 290295 300 Gly Leu Thr Val Tyr Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr305 310 315 320 Ile Arg His Trp Ala Gly His Gln Ile Ser Tyr His Arg ValSer Arg 325 330 335 Gly Ser Asn Leu Gln Gln Met Tyr Gly Thr Asn Gln AsnLeu His Ser 340 345 350 Thr Ser Thr Phe Asp Phe Thr Asn Tyr Asp Ile TyrLys Thr Leu Ser 355 360 365 Lys Asp Ala Val Leu Leu Asp Ile Val Tyr ProGly Tyr Thr Tyr Ile 370 375 380 Phe Phe Gly Met Pro Glu Val Glu Phe PheMet Val Asn Gln Leu Asn 385 390 395 400 Asn Thr Arg Lys Thr Leu Lys TyrAsn Pro Val Ser Lys Asp Ile Ile 405 410 415 Ala Ser Thr Arg Asp Ser GluLeu Glu Leu Pro Pro Glu Thr Ser Asp 420 425 430 Gln Pro Asn Tyr Glu SerTyr Ser His Arg Leu Cys His Ile Thr Ser 435 440 445 Ile Pro Ala Thr GlyAsn Thr Thr Gly Leu Val Pro Val Phe Ser Trp 450 455 460 Thr His Arg SerAla Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile 465 470 475 480 Thr GlnIle Pro Ala Val Lys Cys Trp Asp Asn Leu Pro Phe Val Pro 485 490 495 ValVal Lys Gly Pro Gly His Thr Gly Gly Asp Leu Leu Gln Tyr Asn 500 505 510Arg Ser Thr Gly Ser Val Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu 515 520525 Ala Leu Glu Lys Ala Gly Lys Tyr Arg Val Arg Leu Arg Tyr Ala Thr 530535 540 Asp Ala Asp Ile Val Leu His Val Asn Asp Ala Gln Ile Gln Met Pro545 550 555 560 Lys Thr Met Asn Pro Gly Glu Asp Leu Thr Ser Lys Thr PheLys Val 565 570 575 Ala Asp Ala Ile Thr Thr Leu Asn Leu Ala Thr Asp SerSer Leu Ala 580 585 590 Leu Lys His Asn Leu Gly Glu Asp Pro Asn Ser ThrLeu Ser Gly Ile 595 600 605 Val Tyr Val Asp Arg Ile Glu Phe Ile Pro ValAsp 610 615 620 <210> SEQ ID NO 43 <211> LENGTH: 2013 <212> TYPE: DNA<213> ORGANISM: Bacillus thuringiensis (mutated) <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)...(2013) <221> NAME/KEY: misc_feature<222> LOCATION: (0)...(0) <223> OTHER INFORMATION: LRMS.R1218-1 <400>SEQUENCE: 43 atg agt cca aat aat caa aat gaa tat gaa att ata gat gcg acacct 48 Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 15 10 15 tct act tct gta tcc aat gat tct aac aga tac cct ttt gcg aat gag96 Ser Thr Ser Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 2530 cca aca aat gcg cta caa aat atg gat tat aaa gat tat tta aaa atg 144Pro Thr Asn Ala Leu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45tct gcg gga aat gct agt gaa tac cct ggt tca cct gaa gta ctt gtt 192 SerAla Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 50 55 60 agcgga caa gat gca gct aag gcc gca att gat ata gta ggt aaa tta 240 Ser GlyGln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly Lys Leu 65 70 75 80 ctatca ggt tta ggg gtc cca ttt gtt ggg ccg ata gtg agt ctt tat 288 Leu SerGly Leu Gly Val Pro Phe Val Gly Pro Ile Val Ser Leu Tyr 85 90 95 act caactt att gat att ctg tgg cct tca ggg gaa aag agt caa tgg 336 Thr Gln LeuIle Asp Ile Leu Trp Pro Ser Gly Glu Lys Ser Gln Trp 100 105 110 gaa attttt atg gaa caa gta gaa gaa ctc att aat caa aaa ata gca 384 Glu Ile PheMet Glu Gln Val Glu Glu Leu Ile Asn Gln Lys Ile Ala 115 120 125 gaa tatgca agg aat aaa gcg ctt tcg gaa tta gaa gga tta ggt aat 432 Glu Tyr AlaArg Asn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly Asn 130 135 140 aat taccaa tta tat cta act gcg ctt gaa gaa tgg gaa gaa aat cca 480 Asn Tyr GlnLeu Tyr Leu Thr Ala Leu Glu Glu Trp Glu Glu Asn Pro 145 150 155 160 ttaaga atg tct aga gcc tta cga gat gtg cga aat cga ttt gaa atc 528 Leu ArgMet Ser Arg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu Ile 165 170 175 ctggat agt tta ttt acg caa tat atg cca tct ttt aga gtg aca aat 576 Leu AspSer Leu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr Asn 180 185 190 tttgaa gta cca ttc ctt act gta tat gca atg gca gcc aac ctt cat 624 Phe GluVal Pro Phe Leu Thr Val Tyr Ala Met Ala Ala Asn Leu His 195 200 205 ttactg tta tta aag gac gcg tca att ttt gga gaa gaa tgg gga tgg 672 Leu LeuLeu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Trp 210 215 220 tcaaca act act att aat aac tat tat gat cgt caa atg aaa ctt act 720 Ser ThrThr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu Thr 225 230 235 240gca gaa tat tct gat cac tgt gta aag tgg tat gaa act ggt tta gca 768 AlaGlu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu Ala 245 250 255aaa tta aaa ggc acg agc gct aaa caa tgg gtt gac tat aac caa ttc 816 LysLeu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr Asn Gln Phe 260 265 270cgt aga gaa atg aca ctg gcg gtt tta gat gtt gtt gca tta ttc cca 864 ArgArg Glu Met Thr Leu Ala Val Leu Asp Val Val Ala Leu Phe Pro 275 280 285aat tat gac aca cgc acg tac cca atg gaa acg aaa gca caa cta aca 912 AsnTyr Asp Thr Arg Thr Tyr Pro Met Glu Thr Lys Ala Gln Leu Thr 290 295 300agg gaa gta tat aca gat cca ctg ggc gcg gta aac gtg tct tca att 960 ArgGlu Val Tyr Thr Asp Pro Leu Gly Ala Val Asn Val Ser Ser Ile 305 310 315320 ggt tcc tgg tat gac aaa gca cct tct ttc gga gtg ata gaa tca tcc 1008Gly Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val Ile Glu Ser Ser 325 330335 gtt att cga cca ccc cat gta ttt gat tat ata acg gga ctc aca gtg 1056Val Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr Gly Leu Thr Val 340 345350 tat aca caa tca aga agc att tct tcc gct cgc tat ata aga cat tgg 1104Tyr Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr Ile Arg His Trp 355 360365 gct ggt cat caa ata agc tac cat cgt gtc agt agg ggt agt aat ctt 1152Ala Gly His Gln Ile Ser Tyr His Arg Val Ser Arg Gly Ser Asn Leu 370 375380 caa caa atg tat gga act aat caa aat cta cac agc act agt acc ttt 1200Gln Gln Met Tyr Gly Thr Asn Gln Asn Leu His Ser Thr Ser Thr Phe 385 390395 400 gat ttt acg aat tat gat att tac aag act cta tca aag gat gca gta1248 Asp Phe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala Val 405410 415 ctc ctt gat att gtt tac cct ggt tat acg tat ata ttt ttt gga atg1296 Leu Leu Asp Ile Val Tyr Pro Gly Tyr Thr Tyr Ile Phe Phe Gly Met 420425 430 cca gaa gtc gag ttt ttc atg gta aac caa ttg aat aat acc aga aag1344 Pro Glu Val Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg Lys 435440 445 acg tta aag tat aat cca gtt tcc aaa gat att ata gcg agt aca aga1392 Thr Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Ser Thr Arg 450455 460 gat tcg gaa tta gaa tta cct cca gaa act tca gat caa cca aat tat1440 Asp Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn Tyr 465470 475 480 gag tca tat agc cat aga tta tgt cat atc aca agt att ccc gcgacg 1488 Glu Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala Thr485 490 495 ggt aac act acc gga tta gta cct gta ttt tct tgg aca cat cgaagt 1536 Gly Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr His Arg Ser500 505 510 gca gat tta aac aat aca ata tat tca gat aaa atc act caa attccg 1584 Ala Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile Thr Gln Ile Pro515 520 525 gcc gtt aaa tgt tgg gat aat tta ccg ttt gtt cca gtg gta aaagga 1632 Ala Val Lys Cys Trp Asp Asn Leu Pro Phe Val Pro Val Val Lys Gly530 535 540 cca gga cat aca gga ggg gat tta tta cag tat aat aga agt actggt 1680 Pro Gly His Thr Gly Gly Asp Leu Leu Gln Tyr Asn Arg Ser Thr Gly545 550 555 560 tct gta gga acc tta ttt cta gct cga tat ggc cta gca ttagaa aaa 1728 Ser Val Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu Ala Leu GluLys 565 570 575 gca ggg aaa tat cgt gta aga ctg aga tat gct act gat gcagat att 1776 Ala Gly Lys Tyr Arg Val Arg Leu Arg Tyr Ala Thr Asp Ala AspIle 580 585 590 gta ttg cat gta aac gat gct cag att cag atg cca aaa acaatg aac 1824 Val Leu His Val Asn Asp Ala Gln Ile Gln Met Pro Lys Thr MetAsn 595 600 605 cca ggt gag gat ctg aca tct aaa act ttt aaa gtt gca gatgct atc 1872 Pro Gly Glu Asp Leu Thr Ser Lys Thr Phe Lys Val Ala Asp AlaIle 610 615 620 aca aca tta aat tta gca aca gat agt tcg cta gca ttg aaacat aat 1920 Thr Thr Leu Asn Leu Ala Thr Asp Ser Ser Leu Ala Leu Lys HisAsn 625 630 635 640 tta ggt gaa gac cct aat tca aca tta tct ggt ata gtttac gtt gac 1968 Leu Gly Glu Asp Pro Asn Ser Thr Leu Ser Gly Ile Val TyrVal Asp 645 650 655 cga atc gaa ttc atc cca gta gat gag aca tat gaa gcggaa taa 2013 Arg Ile Glu Phe Ile Pro Val Asp Glu Thr Tyr Glu Ala Glu *660 665 670 <210> SEQ ID NO 44 <211> LENGTH: 670 <212> TYPE: PRT <213>ORGANISM: Bacillus thuringiensis (mutated) <400> SEQUENCE: 44 Met SerPro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 SerThr Ser Val Ser Asn Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 ProThr Asn Ala Leu Gln Asn Met Asp Tyr Lys Asp Tyr Leu Lys Met 35 40 45 SerAla Gly Asn Ala Ser Glu Tyr Pro Gly Ser Pro Glu Val Leu Val 50 55 60 SerGly Gln Asp Ala Ala Lys Ala Ala Ile Asp Ile Val Gly Lys Leu 65 70 75 80Leu Ser Gly Leu Gly Val Pro Phe Val Gly Pro Ile Val Ser Leu Tyr 85 90 95Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly Glu Lys Ser Gln Trp 100 105110 Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile Asn Gln Lys Ile Ala 115120 125 Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly Asn130 135 140 Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu Trp Glu Glu AsnPro 145 150 155 160 Leu Arg Met Ser Arg Ala Leu Arg Asp Val Arg Asn ArgPhe Glu Ile 165 170 175 Leu Asp Ser Leu Phe Thr Gln Tyr Met Pro Ser PheArg Val Thr Asn 180 185 190 Phe Glu Val Pro Phe Leu Thr Val Tyr Ala MetAla Ala Asn Leu His 195 200 205 Leu Leu Leu Leu Lys Asp Ala Ser Ile PheGly Glu Glu Trp Gly Trp 210 215 220 Ser Thr Thr Thr Ile Asn Asn Tyr TyrAsp Arg Gln Met Lys Leu Thr 225 230 235 240 Ala Glu Tyr Ser Asp His CysVal Lys Trp Tyr Glu Thr Gly Leu Ala 245 250 255 Lys Leu Lys Gly Thr SerAla Lys Gln Trp Val Asp Tyr Asn Gln Phe 260 265 270 Arg Arg Glu Met ThrLeu Ala Val Leu Asp Val Val Ala Leu Phe Pro 275 280 285 Asn Tyr Asp ThrArg Thr Tyr Pro Met Glu Thr Lys Ala Gln Leu Thr 290 295 300 Arg Glu ValTyr Thr Asp Pro Leu Gly Ala Val Asn Val Ser Ser Ile 305 310 315 320 GlySer Trp Tyr Asp Lys Ala Pro Ser Phe Gly Val Ile Glu Ser Ser 325 330 335Val Ile Arg Pro Pro His Val Phe Asp Tyr Ile Thr Gly Leu Thr Val 340 345350 Tyr Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg Tyr Ile Arg His Trp 355360 365 Ala Gly His Gln Ile Ser Tyr His Arg Val Ser Arg Gly Ser Asn Leu370 375 380 Gln Gln Met Tyr Gly Thr Asn Gln Asn Leu His Ser Thr Ser ThrPhe 385 390 395 400 Asp Phe Thr Asn Tyr Asp Ile Tyr Lys Thr Leu Ser LysAsp Ala Val 405 410 415 Leu Leu Asp Ile Val Tyr Pro Gly Tyr Thr Tyr IlePhe Phe Gly Met 420 425 430 Pro Glu Val Glu Phe Phe Met Val Asn Gln LeuAsn Asn Thr Arg Lys 435 440 445 Thr Leu Lys Tyr Asn Pro Val Ser Lys AspIle Ile Ala Ser Thr Arg 450 455 460 Asp Ser Glu Leu Glu Leu Pro Pro GluThr Ser Asp Gln Pro Asn Tyr 465 470 475 480 Glu Ser Tyr Ser His Arg LeuCys His Ile Thr Ser Ile Pro Ala Thr 485 490 495 Gly Asn Thr Thr Gly LeuVal Pro Val Phe Ser Trp Thr His Arg Ser 500 505 510 Ala Asp Leu Asn AsnThr Ile Tyr Ser Asp Lys Ile Thr Gln Ile Pro 515 520 525 Ala Val Lys CysTrp Asp Asn Leu Pro Phe Val Pro Val Val Lys Gly 530 535 540 Pro Gly HisThr Gly Gly Asp Leu Leu Gln Tyr Asn Arg Ser Thr Gly 545 550 555 560 SerVal Gly Thr Leu Phe Leu Ala Arg Tyr Gly Leu Ala Leu Glu Lys 565 570 575Ala Gly Lys Tyr Arg Val Arg Leu Arg Tyr Ala Thr Asp Ala Asp Ile 580 585590 Val Leu His Val Asn Asp Ala Gln Ile Gln Met Pro Lys Thr Met Asn 595600 605 Pro Gly Glu Asp Leu Thr Ser Lys Thr Phe Lys Val Ala Asp Ala Ile610 615 620 Thr Thr Leu Asn Leu Ala Thr Asp Ser Ser Leu Ala Leu Lys HisAsn 625 630 635 640 Leu Gly Glu Asp Pro Asn Ser Thr Leu Ser Gly Ile ValTyr Val Asp 645 650 655 Arg Ile Glu Phe Ile Pro Val Asp Glu Thr Tyr GluAla Glu 660 665 670 <210> SEQ ID NO 45 <211> LENGTH: 1854 <212> TYPE:DNA <213> ORGANISM: Bacillus thuringiensis (mutated) <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(1854) <221> NAME/KEY:misc_feature <222> LOCATION: (0)...(0) <223> OTHER INFORMATION:LRMS.R49PVD <400> SEQUENCE: 45 atg tct gcg gga aat gct agt gaa tac cctggt tca cct gaa gta ctt 48 Met Ser Ala Gly Asn Ala Ser Glu Tyr Pro GlySer Pro Glu Val Leu 1 5 10 15 gtt agc gga caa gat gca gct aag gcc gcaatt gat ata gta ggt aaa 96 Val Ser Gly Gln Asp Ala Ala Lys Ala Ala IleAsp Ile Val Gly Lys 20 25 30 tta cta tca ggt tta ggg gtc cca ttt gtt gggccg ata gtg agt ctt 144 Leu Leu Ser Gly Leu Gly Val Pro Phe Val Gly ProIle Val Ser Leu 35 40 45 tat act caa ctt att gat att ctg tgg cct tca ggggaa aag agt caa 192 Tyr Thr Gln Leu Ile Asp Ile Leu Trp Pro Ser Gly GluLys Ser Gln 50 55 60 tgg gaa att ttt atg gaa caa gta gaa gaa ctc att aatcaa aaa ata 240 Trp Glu Ile Phe Met Glu Gln Val Glu Glu Leu Ile Asn GlnLys Ile 65 70 75 80 gca gaa tat gca agg aat aaa gcg ctt tcg gaa tta gaagga tta ggt 288 Ala Glu Tyr Ala Arg Asn Lys Ala Leu Ser Glu Leu Glu GlyLeu Gly 85 90 95 aat aat tac caa tta tat cta act gcg ctt gaa gaa tgg gaagaa aat 336 Asn Asn Tyr Gln Leu Tyr Leu Thr Ala Leu Glu Glu Trp Glu GluAsn 100 105 110 cca tta aga atg tct aga gcc tta cga gat gtg cga aat cgattt gaa 384 Pro Leu Arg Met Ser Arg Ala Leu Arg Asp Val Arg Asn Arg PheGlu 115 120 125 atc ctg gat agt tta ttt acg caa tat atg cca tct ttt agagtg aca 432 Ile Leu Asp Ser Leu Phe Thr Gln Tyr Met Pro Ser Phe Arg ValThr 130 135 140 aat ttt gaa gta cca ttc ctt act gta tat gca atg gca gccaac ctt 480 Asn Phe Glu Val Pro Phe Leu Thr Val Tyr Ala Met Ala Ala AsnLeu 145 150 155 160 cat tta ctg tta tta aag gac gcg tca att ttt gga gaagaa tgg gga 528 His Leu Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu GluTrp Gly 165 170 175 tgg tca aca act act att aat aac tat tat gat cgt caaatg aaa ctt 576 Trp Ser Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln MetLys Leu 180 185 190 act gca gaa tat tct gat cac tgt gta aag tgg tat gaaact ggt tta 624 Thr Ala Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu ThrGly Leu 195 200 205 gca aaa tta aaa ggc acg agc gct aaa caa tgg gtt gactat aac caa 672 Ala Lys Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp TyrAsn Gln 210 215 220 ttc cgt aga gaa atg aca ctg gcg gtt tta gat gtt gttgca tta ttc 720 Phe Arg Arg Glu Met Thr Leu Ala Val Leu Asp Val Val AlaLeu Phe 225 230 235 240 cca aat tat gac aca cgc acg tac cca atg gaa acgaaa gca caa cta 768 Pro Asn Tyr Asp Thr Arg Thr Tyr Pro Met Glu Thr LysAla Gln Leu 245 250 255 aca agg gaa gta tat aca gat cca ctg ggc gcg gtaaac gtg tct tca 816 Thr Arg Glu Val Tyr Thr Asp Pro Leu Gly Ala Val AsnVal Ser Ser 260 265 270 att ggt tcc tgg tat gac aaa gca cct tct ttc ggagtg ata gaa tca 864 Ile Gly Ser Trp Tyr Asp Lys Ala Pro Ser Phe Gly ValIle Glu Ser 275 280 285 tcc gtt att cga cca ccc cat gta ttt gat tat ataacg gga ctc aca 912 Ser Val Ile Arg Pro Pro His Val Phe Asp Tyr Ile ThrGly Leu Thr 290 295 300 gtg tat aca caa tca aga agc att tct tcc gct cgctat ata aga cat 960 Val Tyr Thr Gln Ser Arg Ser Ile Ser Ser Ala Arg TyrIle Arg His 305 310 315 320 tgg gct ggt cat caa ata agc tac cat cgt gtcagt agg ggt agt aat 1008 Trp Ala Gly His Gln Ile Ser Tyr His Arg Val SerArg Gly Ser Asn 325 330 335 ctt caa caa atg tat gga act aat caa aat ctacac agc act agt acc 1056 Leu Gln Gln Met Tyr Gly Thr Asn Gln Asn Leu HisSer Thr Ser Thr 340 345 350 ttt gat ttt acg aat tat gat att tac aag actcta tca aag gat gca 1104 Phe Asp Phe Thr Asn Tyr Asp Ile Tyr Lys Thr LeuSer Lys Asp Ala 355 360 365 gta ctc ctt gat att gtt tac cct ggt tat acgtat ata ttt ttt gga 1152 Val Leu Leu Asp Ile Val Tyr Pro Gly Tyr Thr TyrIle Phe Phe Gly 370 375 380 atg cca gaa gtc gag ttt ttc atg gta aac caattg aat aat acc aga 1200 Met Pro Glu Val Glu Phe Phe Met Val Asn Gln LeuAsn Asn Thr Arg 385 390 395 400 aag acg tta aag tat aat cca gtt tcc aaagat att ata gcg agt aca 1248 Lys Thr Leu Lys Tyr Asn Pro Val Ser Lys AspIle Ile Ala Ser Thr 405 410 415 aga gat tcg gaa tta gaa tta cct cca gaaact tca gat caa cca aat 1296 Arg Asp Ser Glu Leu Glu Leu Pro Pro Glu ThrSer Asp Gln Pro Asn 420 425 430 tat gag tca tat agc cat aga tta tgt catatc aca agt att ccc gcg 1344 Tyr Glu Ser Tyr Ser His Arg Leu Cys His IleThr Ser Ile Pro Ala 435 440 445 acg ggt aac act acc gga tta gta cct gtattt tct tgg aca cat cga 1392 Thr Gly Asn Thr Thr Gly Leu Val Pro Val PheSer Trp Thr His Arg 450 455 460 agt gca gat tta aac aat aca ata tat tcagat aaa atc act caa att 1440 Ser Ala Asp Leu Asn Asn Thr Ile Tyr Ser AspLys Ile Thr Gln Ile 465 470 475 480 ccg gcc gtt aaa tgt tgg gat aat ttaccg ttt gtt cca gtg gta aaa 1488 Pro Ala Val Lys Cys Trp Asp Asn Leu ProPhe Val Pro Val Val Lys 485 490 495 gga cca gga cat aca gga ggg gat ttatta cag tat aat aga agt act 1536 Gly Pro Gly His Thr Gly Gly Asp Leu LeuGln Tyr Asn Arg Ser Thr 500 505 510 ggt tct gta gga acc tta ttt cta gctcga tat ggc cta gca tta gaa 1584 Gly Ser Val Gly Thr Leu Phe Leu Ala ArgTyr Gly Leu Ala Leu Glu 515 520 525 aaa gca ggg aaa tat cgt gta aga ctgaga tat gct act gat gca gat 1632 Lys Ala Gly Lys Tyr Arg Val Arg Leu ArgTyr Ala Thr Asp Ala Asp 530 535 540 att gta ttg cat gta aac gat gct cagatt cag atg cca aaa aca atg 1680 Ile Val Leu His Val Asn Asp Ala Gln IleGln Met Pro Lys Thr Met 545 550 555 560 aac cca ggt gag gat ctg aca tctaaa act ttt aaa gtt gca gat gct 1728 Asn Pro Gly Glu Asp Leu Thr Ser LysThr Phe Lys Val Ala Asp Ala 565 570 575 atc aca aca tta aat tta gca acagat agt tcg cta gca ttg aaa cat 1776 Ile Thr Thr Leu Asn Leu Ala Thr AspSer Ser Leu Ala Leu Lys His 580 585 590 aat tta ggt gaa gac cct aat tcaaca tta tct ggt ata gtt tac gtt 1824 Asn Leu Gly Glu Asp Pro Asn Ser ThrLeu Ser Gly Ile Val Tyr Val 595 600 605 gac cga atc gaa ttc atc cca gtagat taa 1854 Asp Arg Ile Glu Phe Ile Pro Val Asp * 610 615 <210> SEQ IDNO 46 <211> LENGTH: 617 <212> TYPE: PRT <213> ORGANISM: Bacillusthuringiensis (mutated) <400> SEQUENCE: 46 Met Ser Ala Gly Asn Ala SerGlu Tyr Pro Gly Ser Pro Glu Val Leu 1 5 10 15 Val Ser Gly Gln Asp AlaAla Lys Ala Ala Ile Asp Ile Val Gly Lys 20 25 30 Leu Leu Ser Gly Leu GlyVal Pro Phe Val Gly Pro Ile Val Ser Leu 35 40 45 Tyr Thr Gln Leu Ile AspIle Leu Trp Pro Ser Gly Glu Lys Ser Gln 50 55 60 Trp Glu Ile Phe Met GluGln Val Glu Glu Leu Ile Asn Gln Lys Ile 65 70 75 80 Ala Glu Tyr Ala ArgAsn Lys Ala Leu Ser Glu Leu Glu Gly Leu Gly 85 90 95 Asn Asn Tyr Gln LeuTyr Leu Thr Ala Leu Glu Glu Trp Glu Glu Asn 100 105 110 Pro Leu Arg MetSer Arg Ala Leu Arg Asp Val Arg Asn Arg Phe Glu 115 120 125 Ile Leu AspSer Leu Phe Thr Gln Tyr Met Pro Ser Phe Arg Val Thr 130 135 140 Asn PheGlu Val Pro Phe Leu Thr Val Tyr Ala Met Ala Ala Asn Leu 145 150 155 160His Leu Leu Leu Leu Lys Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly 165 170175 Trp Ser Thr Thr Thr Ile Asn Asn Tyr Tyr Asp Arg Gln Met Lys Leu 180185 190 Thr Ala Glu Tyr Ser Asp His Cys Val Lys Trp Tyr Glu Thr Gly Leu195 200 205 Ala Lys Leu Lys Gly Thr Ser Ala Lys Gln Trp Val Asp Tyr AsnGln 210 215 220 Phe Arg Arg Glu Met Thr Leu Ala Val Leu Asp Val Val AlaLeu Phe 225 230 235 240 Pro Asn Tyr Asp Thr Arg Thr Tyr Pro Met Glu ThrLys Ala Gln Leu 245 250 255 Thr Arg Glu Val Tyr Thr Asp Pro Leu Gly AlaVal Asn Val Ser Ser 260 265 270 Ile Gly Ser Trp Tyr Asp Lys Ala Pro SerPhe Gly Val Ile Glu Ser 275 280 285 Ser Val Ile Arg Pro Pro His Val PheAsp Tyr Ile Thr Gly Leu Thr 290 295 300 Val Tyr Thr Gln Ser Arg Ser IleSer Ser Ala Arg Tyr Ile Arg His 305 310 315 320 Trp Ala Gly His Gln IleSer Tyr His Arg Val Ser Arg Gly Ser Asn 325 330 335 Leu Gln Gln Met TyrGly Thr Asn Gln Asn Leu His Ser Thr Ser Thr 340 345 350 Phe Asp Phe ThrAsn Tyr Asp Ile Tyr Lys Thr Leu Ser Lys Asp Ala 355 360 365 Val Leu LeuAsp Ile Val Tyr Pro Gly Tyr Thr Tyr Ile Phe Phe Gly 370 375 380 Met ProGlu Val Glu Phe Phe Met Val Asn Gln Leu Asn Asn Thr Arg 385 390 395 400Lys Thr Leu Lys Tyr Asn Pro Val Ser Lys Asp Ile Ile Ala Ser Thr 405 410415 Arg Asp Ser Glu Leu Glu Leu Pro Pro Glu Thr Ser Asp Gln Pro Asn 420425 430 Tyr Glu Ser Tyr Ser His Arg Leu Cys His Ile Thr Ser Ile Pro Ala435 440 445 Thr Gly Asn Thr Thr Gly Leu Val Pro Val Phe Ser Trp Thr HisArg 450 455 460 Ser Ala Asp Leu Asn Asn Thr Ile Tyr Ser Asp Lys Ile ThrGln Ile 465 470 475 480 Pro Ala Val Lys Cys Trp Asp Asn Leu Pro Phe ValPro Val Val Lys 485 490 495 Gly Pro Gly His Thr Gly Gly Asp Leu Leu GlnTyr Asn Arg Ser Thr 500 505 510 Gly Ser Val Gly Thr Leu Phe Leu Ala ArgTyr Gly Leu Ala Leu Glu 515 520 525 Lys Ala Gly Lys Tyr Arg Val Arg LeuArg Tyr Ala Thr Asp Ala Asp 530 535 540 Ile Val Leu His Val Asn Asp AlaGln Ile Gln Met Pro Lys Thr Met 545 550 555 560 Asn Pro Gly Glu Asp LeuThr Ser Lys Thr Phe Lys Val Ala Asp Ala 565 570 575 Ile Thr Thr Leu AsnLeu Ala Thr Asp Ser Ser Leu Ala Leu Lys His 580 585 590 Asn Leu Gly GluAsp Pro Asn Ser Thr Leu Ser Gly Ile Val Tyr Val 595 600 605 Asp Arg IleGlu Phe Ile Pro Val Asp 610 615 <210> SEQ ID NO 47 <211> LENGTH: 12<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: LRMS Insert <400> SEQUENCE: 47 ttaagaatgt ct 12 <210>SEQ ID NO 48 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: LRMS Insert <400>SEQUENCE: 48 Leu Arg Met Ser 1

That which is claimed:
 1. An isolated nucleic acid comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 15, or 17; (b)a nucleotide sequence encoding the amino acid sequence set forth in SEQID NO:2, 4, 6, 8, 16,or 18; (c) a nucleotide sequence characterized byat least 88% sequence identity to the nucleotide sequence set forth in(a); (d) a nucleotide sequence encoding a protein comprising an aminoacid sequence characterized by at least 85% sequence identity to theamino acid sequence set forth in (b); (e) an antisense nucleotidesequence corresponding to the nucleotide sequence of any one of (a) to(d); and (f) a nucleotide sequence that hybridizes under stringentconditions to the nucleotide sequence of any one of (a) to (e).
 2. Thenucleic acid according to claim 1, wherein the nucleotide sequence isoptimized for expression in a plant.
 3. An expression cassettecomprising a nucleic acid according to claim 1, wherein said nucleotidesequence is operably linked to a promoter that drives expression in amicroorganism or in a plant cell.
 4. An isolated pesticidal polypeptideselected from group consisting of: (a) a polypeptide comprising an aminoacid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 16, or 18; and (b) apolypeptide characterized by at least 85% sequence identity to the aminoacid sequence of (a).
 5. The polypeptide according to claim 4, whereinsaid polypeptide is characterized by pesticidal activity against atleast one pest belonging to the order Coeleopteran.
 6. A pesticidalcomposition comprising at least one polypeptide according to claim 5 incombination with a carrier.
 7. A method for impacting an insect pestcomprising applying the pesticidal composition according to claim 6 tothe environment of the insect pest by a procedure selected from thegroup consisting of spraying, dusting, broadcasting, and seed coating.8. The method according to claim 7, wherein said insect pest is selectedfrom the group consisting of Colorado potato beetle, western cornrootworm, and southern corn rootworm.
 9. A transformed plant comprisingin its genome at least one stably incorporated nucleotide constructcomprising a coding sequence operably linked to a promoter that drivesexpression of a polypeptide that is pesticidal for at least one pestbelonging to the order Coleopteran, wherein said coding sequence isselected from the group consisting of: (a) a nucleotide sequence setforth in SEQ ID NO:1, 3, 5, 7, 9, 15, or 17; (b) a nucleotide sequenceencoding the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 16,or 18; (c) a nucleotide sequence characterized by at least 88% sequenceidentity to the nucleotide sequence set forth in (a); (d) a nucleotidesequence encoding a protein comprising an amino acid sequencecharacterized by at least 85% sequence identity to the amino acidsequence set forth in (b); (e) a nucleotide sequence according to anyone of (a) to (d) that comprises codons optimized for expression in aplant; (f) an antisense nucleotide sequence corresponding to thenucleotide sequence of any one of (a) to (d); and (g) a nucleotidesequence that hybridizes under stringent conditions to the nucleotidesequence of any one of (a) to (f).
 10. The plant according to claim 9,wherein the plant is a monocot.
 11. The plant according to claim 9,wherein said plant is a dicot.
 12. Transformed seed of the plantaccording to claim
 9. 13. A transformed microorganism comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 15, 17, 27, or28; (b) a nucleotide sequence encoding the amino acid sequence set forthin SEQ ID NO:2, 4, 6, 8, 16, or 18; (c) a nucleotide sequencecharacterized by at least 88% sequence identity to the nucleotidesequence set forth in (a); (d) a nucleotide sequence encoding a proteincomprising an amino acid sequence characterized by at least 85% sequenceidentity to the amino acid sequence set forth in (b); (e) an antisensenucleotide sequence corresponding to the nucleotide sequence of any oneof (a) to (d); and (f) a nucleotide sequence that hybridizes understringent conditions to the nucleotide sequence of any one of (a) to(e).
 14. The transformed microorganism according to claim 13, whereinthe nucleotide sequence is operably linked to a promoter that drivesexpression in said microorganism.
 15. A pesticidal compositioncomprising a transformed microorganism according to claim 13 and acarrier, wherein said composition has been treated to prolong pesticidalactivity.
 16. A method of impacting a pest comprising applying thepesticidal composition according to claim 14 to the environment of thepest by a procedure selected from the group consisting of spraying,dusting, broadcasting, and seed coating.
 17. A method for impacting aplant pest comprising introducing into said plant or cell thereof atleast one nucleotide construct comprising a coding sequence operablylinked to a promoter that drives expression of a pesticidal polypeptidein plant cells, wherein said nucleotide sequence is selected from thegroup consisting of: (a) a nucleotide sequence set forth in SEQ ID NO:1,3, 5, 7, 9, 15, or 17; (b) a nucleotide sequence encoding the amino acidsequence set forth in SEQ ID NO:2, 4, 6, 8, 16, or 18; (c) a nucleotidesequence characterized by at least 88% sequence identity to thenucleotide sequence set forth in (a); (d) a nucleotide sequence encodinga protein comprising an amino acid sequence characterized by at least85% sequence identity to the amino acid sequence set forth in (b); (e)an antisense nucleotide sequence corresponding to the nucleotidesequence of any one of (a) to (d); and (f) a nucleotide sequence thathybridizes under stringent conditions to the nucleotide sequence of anyone of (a) to (e).
 18. The method according to claim 17, wherein theplant produces a polypeptide characterized by pesticidal activityagainst at least one pest of the order Coelopteran.
 19. The methodaccording to claim 18, wherein said plant pest is selected from thegroup consisting of Colorado potato beetle, western corn rootworm, andsouthern corn rootworm.
 20. A variant of the nucleic acid set forth inSEQ ID NO:19 wherein the variant comprises a nucleotide sequence havingat least one additional codon not present in the nucleotide sequence setforth in SEQ ID NO:19, wherein the at least one additional codonintroduces an additional protease-sensitive site in the loop regionbetween alpha-helices 3 and 4 of domain 1 of the encoded polypeptide,and further wherein the polypeptide encoded by the variant ischaracterized by improved pesticidal activity against a pest belongingto the order Coleopteran relative to the activity of the polypeptide setforth in SEQ ID NO:2.
 21. The nucleic acid according to claim 20,wherein the nucleotide sequence is optimized for expression in a plant.22. An expression cassette comprising a nucleic acid according to claim20, wherein said nucleotide sequence is operably linked to a promoterthat drives expression in a microorganism or in a plant cell.
 23. Anisolated nucleic acid comprising a nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence set forth in any one ofSEQ ID NOS: 5, 11, 15, 21, 23, 39, and 43; (b) a nucleotide sequenceencoding any one of the amino acid sequences set forth in SEQ ID NOS: 6,12, 16, 22, 24, 40, and 44; (c) a nucleotide sequence encoding apolypeptide variant of SEQ ID NO:16, wherein the polpeptide comprises anadditional protease-sensitive cleavage site inserted between amino acidresidues 164 and 165 of SEQ ID NO: 16; (d) a nucleotide sequenceencoding a polypeptide variant of SEQ ID NO:16, wherein the polypeptidevariant comprises an additional amino acid sequence designed tointroduce a trypsin cleavage site between amino acid residues 164 and165 of SEQ ID NO: 16; (e) a nucleotide sequence encoding a polypeptidevariant of SEQ ID NO:16, wherein the polypeptide comprises an additionalamino acid sequence designed to introduce a chymotrypsin cleavage sitebetween amino acid residues 160 and 161 of SEQ ID NO: 16; and (f) anucleotide sequence encoding a polypeptide variant of SEQ ID NO:16,wherein the polypeptide comprises an amino acid sequence in whichresidues corresponding to positions 161 through 163 of SEQ ID NO:16 areremoved and additional amino acids comprising a chymotrypsin cleavagesite are in introduced in their place.
 24. The nucleic acid according toclaim 23, wherein the nucleotide sequence is optimized for expression ina plant.
 25. A variant of the nucleic acid set forth in SEQ ID NO: 15,wherein the variant comprises a nucleotide sequence that includes atleast one additional codon that introduces an additionalprotease-sensitive site in the loop region between alpha-helices 3 and 4of domain 1 of the polypeptide encoded by the variant nucleic acid, andfurther wherein the encoded polypeptide is characterized by improvedpesticidal activity against a pest belonging to the order Coleopteranrelative to the activity of the polypeptide set forth in SEQ ID NO: 2.26. An expression cassette comprising a nucleic acid according to claim25, wherein said nucleotide sequence is operably linked to a promoterthat drives expression in a microorganism or in a plant cell.
 27. Anisolated nucleic acid comprising a nucleic acid sequence selected fromthe group consisting of: (a) a nucleotide sequence set forth in SEQ IDNO:19, 29, 31, 33, 41, or 45; (b) a nucleotide sequence encoding theamino acid sequence set forth in SEQ ID NO:20, 30, 32, 34, 42, or 46;(c) a nucleotide sequence comprising a variant of SEQ ID NO: 19, whereinthe variant encodes a polypeptide characterized by an additionalprotease-sensitive cleavage site inserted immediately 5′ to amino acid114 of SEQ ID NO: 20; (d) a nucleotide sequence comprising a variant ofSEQ ID NO:19, wherein the variant encodes a polypeptide characterized byan additional amino acid sequence designed to introduce a trypsincleavage site between amino acid residues 113 and 114 of SEQ ID NO: 20;(e) a nucleotide sequence comprising a variant of SEQ ID NO:19, whereinthe variant encodes a polypeptide characterized by an additional aminoacid sequence designed to introduce a chymotrypsin cleavage site betweenamino acid residues 113 and 114 of SEQ ID NO:20; and (f) a nucleotidesequence comprising a variant of SEQ ID NO:19, wherein the variantencodes a polypeptide characterized by an amino acid sequence in whichthe amino acids located at positions 114 through 116 of SEQ ID NO:20 areremoved and additional amino acids comprising a chymotrypsin site areinserted in their place.
 28. The nucleic acid according to claim 27,wherein the nucleotide sequence is optimized for expression in a plant.29. A variant of the polypeptide set forth in SEQ ID NO: 16, wherein thevariant comprises an amino acid sequence that includes at least oneadditional amino acid residue that introduces an additionalprotease-sensitive site in the loop region between alpha-helices 3 and 4of domain 1 of the polypeptide, and further wherein the encodedpolypeptide is characterized by improved pesticidal activity against apest belonging to the order Coleopteran relative to the activity of thepolypeptide set forth in SEQ ID NO:2.
 30. An isolated pesticidalpolypeptide comprising an amino acid sequence set forth in SEQ ID NOS:6,12, 16, 20, 22, 24, 30, 32, 34, 40, 42, 44, or
 46. 31. A transformedplant comprising in its genome at least one stably incorporatednucleotide construct comprising a coding sequence operably linked to apromoter that drives expression of a pesticidal polypeptide in cells oftransformed plant, wherein said coding sequence is selected from thegroup consisting of: (a) a nucleotide sequence set forth in SEQ IDNOS:11, 19, 21, 23, 29, 31, 33, 39, 41, 43, or 45; (b) a nucleotidesequence encoding the amino acid sequence set forth in SEQ ID NO:12, 20,22, 24, 30, 32, 34, 40, 42, 44, or 46; (c) a nucleotide sequenceencoding a polypeptide variant of SEQ ID NO:16, wherein the variantcomprises an additional protease-sensitive cleavage site insertedbetween amino acid residues 164 and 165 of SEQ ID NO:16; (d) anucleotide sequence encoding a polypeptide variant of SEQ ID NO:16,wherein the variant comprises an additional amino acid sequence designedto introduce a trypsin cleavage site between amino acid residues 164 and165 of SEQ ID NO:16; (e) a nucleotide sequence encoding a polypeptidevariant of SEQ ID NO:16, wherein the variant comprises an additionalamino acid sequence designed to introduce a chymotrypsin cleavage sitebetween amino acid residues 160 and 161 of SEQ ID NO: 16; (f) anucleotide sequence encoding a polypeptide variant of SEQ ID NO:16,wherein amino acid residues corresponding to amino acids 161 through 163of SEQ ID NO:16 are removed and amino acids comprising a chymotrypsincleavage site are introduced in their place; (g) a nucleotide sequencecomprising a variant of SEQ ID NO:19, wherein the variant encodes apolypeptide characterized by an additional protease-sensitive cleavagesite immediately 5′ to amino acid 114 of SEQ ID NO: 20; (h) a nucleotidesequence comprising a variant of SEQ ID NO:19, wherein the variantencodes a polypeptide characterized by an additional amino acid selectedto introduce a trypsin cleavage site immediately 5′ of amino acid 114 ofSEQ ID NO: 20; i) a nucleotide sequence comprising a variant of SEQ IDNO:19, wherein the variant comprises additional nucleic acid residuesdesigned to introduce a chymotrypsin cleavage site between amino acidresidues 113 and 114 of SEQ ID NO: 20; j) a nucleotide sequence encodinga polypeptide variant of SEQ ID NO:20, wherein the amino acid residueslocated at positions 114 through 116 of SEQ ID NO:20 are removed andadditional amino acids comprising a chymotrypsin site are in theirplace; and k) a nucleotide sequence according to any one of (a) to (j)that comprises codons optimized for expression in a plant.
 32. Thetransformed plant according to claim 31, wherein cells of thetransformed plant express a polypeptide that is pesticidal for at leastone pest belonging to the order Coleopteran.
 33. The plant according toclaim 32, wherein said plant is a monocot.
 34. The plant according toclaim 32, wherein said plant is a dicot.
 35. Transformed seed of theplant according to claim
 32. 36. A transformed microorganism comprisinga nucleic acid selected from the group consisting of: (a) a nucleotidesequence set forth in SEQ ID NO:11, 19, 21, 23, 29, 31, 33, 39, 41, 43,or 45; (b) a nucleotide sequence encoding the amino acid sequence setforth in SEQ ID NO:12, 20, 22, 24, 30, 32, 34, 40, 42, 44, or 46; (c) anucleotide sequence encoding a variant of SEQ ID NO:19, wherein thevariant comprises a nucleic acid insert designed to introduce anadditional protease-sensitive site between amino acid residues 117 and118 of SEQ ID NO: 20; and (d) a nucleotide sequence encoding apolypeptide variant of SEQ ID NO:16, wherein the variant comprises anadditional protease-sensitive site inserted between amino acid residues164 and 165 of SEQ ID NO:16; and (e) a nucleotide sequence encoding apolypeptide variant of SEQ ID NO:20, wherein the variant comprises anadditional protease-sensitive site inserted between amino acid residues117 and 118 of SEQ ID NO:20.